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Two papers in this issue of Molecular Microbiology from Carol Gross and colleagues describe experiments that investigate how two alternative Escherichia coliσ proteins recognize target promoter −10 regions. A combination of genetics, biochemistry and bioinformatics is used to show that determinants in two adjacent domains of the σ proteins contact discrete sequence elements in the −10 regions. [ABSTRACT FROM AUTHOR]

Heme, a Fe-centred hexacoordinated organometallic compound mainly present in blood proteins, acts as a major source of iron (Fe2+) for various hemolytic microorganisms. This makes blood an essential mediumsupplement for characterizing such organisms. Considering the increasing blood demand for this purpose, we are proposing an alternative approach for hemolytic characterization of bacteria using plant derivative. For the first time, we present the kinetic model of growth of Staphylococcus aureus, a beta-hemolytic, gram-positive rod-shaped bacterium, and subsequent heme uptake at an optimized concentration of crude proteins of Beta vulgaris (red beetroot). In this paper, we have determined the heme content of the beetroot juice and demonstrated the ability of S. aureus to uptake heme from beetroot protein solution. According to the determined growth kinetics, the doubling time of the bacterium in the beetroot protein supplemented medium was 25 minutes. The heme uptake analysis showed a zeroth order kinetics with a constant decline rate of 0.19 μM h-1. [ABSTRACT FROM AUTHOR]

The Min protein system creates a dynamic spatial pattern in Escherichia coli cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution. [ABSTRACT FROM AUTHOR]

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations. [ABSTRACT FROM AUTHOR]

Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5′UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5′UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels. [ABSTRACT FROM AUTHOR]

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (Km) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation. [ABSTRACT FROM AUTHOR]

In the present study, silver nanoparticles (AgNPs) were synthesised by adding 1 mM Ag nitrate solution to different concentrations (1%, 2.5%, 5%) of branch extracts of Eurycoma longifolia, a well known medicinal plant in South–East Asian countries. Characterisation of AgNPs was carried out using techniques such as ultraviolet–visible spectrophotometry, X‐ray diffractrometry, Fourier transform infrared–attenuated total reflection spectroscopy (FTIR–ATR), scanning electron microscopy. XRD analysis revealed face centre cubic structure of AgNPs and FTIR–ATR showed that primary and secondary amide groups in combination with the protein molecules present in the branch extract were responsible for the reduction and stabilisation of AgNPs. Furthermore, antioxidant [2,2‐diphenyl‐1‐picrylhydrazyl and 2,2′‐Azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid)], antimicrobial and anticancer activities of AgNPs were investigated. The highest bactericidal activity of these biogenic AgNPs was found against Escherichia coli with zone inhibition of 11 mm. AgNPs exhibited significant anticancer activity against human glioma cells (DBTRG and U87) and human breast adenocarcinoma cells (MCF‐7 and MDA‐MB‐231) with IC50 values of 33, 42, 60 and 38 µg/ml. [ABSTRACT FROM AUTHOR]

The primary structure of protein S8 from the 30-S ribosomal subunit of Escherichia coil was determined mainly by automatic Edman degradation using a modified Beckman protein sequenator and the solid-phase sequenator of Laursen. The complete sequence, containing 109 amino acids, was derived by analysing peptides from tryptic, chymotryptic, thermolysin, staphylococcal protease and cyanogen bromide digestion of the protein. The amino acid composition was found to be (aspartic acid)6, (asparagine)3, (threonine)5, (serine)5, (glutamic acid)7, (glutamine)6, (proline)5, (glycine)6, (alanine)11, (valine)9, (methionine)4, (isoleucine)7, (leucine)9, (tyrosine)3, (phenylalanine)3, (lysine)11, (arginine)8, (cysteine)1. S8 is a basic protein and binds to the 16-S RNA; knowledge of its sequence is necessary for a detailed study of its interaction with the ribosomal RNA. [ABSTRACT FROM AUTHOR]

The present study reports the synthesis of silver nanoparticles (AgNPs) using both biological and chemical routes to find out the best method for control of their size and activity. The fungal agent (Fusarium oxysporum) and the plant (Azadirachta indica) were found to be the best source for AgNPs synthesis. Both biosynthesis and chemosynthesis were achieved by challenging filtrate with AgNO3 (1 mM) solution. The synthesised nanoparticles were characterised by ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, nanoparticle tracking analysis (LM20), zeta potential measurement and transmission electron microscopy. The biologically synthesised nanoparticles were spherical, polydispersed and in the range of 10–40 nm, while chemically synthesised nanoparticles were highly monodispersed with a size of 5 nm. The antimicrobial assay against Escherichia coli and Staphylococcus aureus proved biogenic AgNPs to be more potent antibacterial agents than chemically synthesised AgNPs. The possible antibacterial mechanism of AgNPs has also been discussed. Biogenic AgNPs have shown more activity because of the protein capping and their mode of entry into the bacterial cell. These findings may encourage the use of biosynthesis over the chemosynthesis method. [ABSTRACT FROM AUTHOR]

Previously, the role of YidC in the membrane protein biogenesis of the F0 sector of the Escherichia coli F1F0 ATP synthase was investigated. Whereas subunits a and c of the F1F0 ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537–10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F0 sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH2-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway. [ABSTRACT FROM AUTHOR]

Solution structure of the region 4 of σ[sup70] subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain [sup13]C resonances for a recombinant Rise-tagged protein containing the whole region 4 and a part of region 3.2 of σ[sup70] in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the [sup13]Cα, [sup13]CO, and [sup1]Hα nuclei and of vicinal [sup3]J[subHNHα] coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and [sup3]J[subHNHα] couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the σ[supA] subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and σ[sup70] of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R[sub1] and R[sub2], and {[sup1]H}-[sup15]N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of σ[sup70][sub4] protein in its protonated state. [ABSTRACT FROM AUTHOR]

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms α, β and γ, differenciated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive β-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gone for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by λ540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1bx is the first membrane-bound form of pbp 1b able to bind labelled β-lactams, and is the precursor of pbp 1bβ which is, in turn, the precursor of pbp 1bγ. [ABSTRACT FROM AUTHOR]

The accuracy of Qβ translation by Escherichia coli extracts in polymix and a conventional Tris/Mg2+ system has been followed. Misinsertions of histidine and of tryptophan into the phage coat protein were less frequent in polymix than in Tris/Mg2+, as were errors leading to u change in the coat protein pI. Even the lowest Qβ error rates, however, were still an order of magnitude greater than those for poly(U) or poly(U-G) translation. Comparing Qβ translational errors made in vitro to those found in whole cells, histidine misinsertions were almost twice as frequent, errors leading to a coat protein charge change six times more frequent and tryptophan misinsertions at least 15 times more frequent in vitro. The relation of these findings to measurements of translational accuracy and to factors affecting fidelity is discussed. [ABSTRACT FROM AUTHOR]

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at position 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study. [ABSTRACT FROM AUTHOR]

FadR, an acylCoA-dependent Escherichia coli transcription factor controlling the expression of genes involved in fatty-acid degradation and synthesis, has been crystallized. Crystals of two binary complexes were obtained. The FadR-CoA complex crystallized in space group C2221, with unit-cell parameters a = 61.3, b = 102.0, c = 91.3 Å. The FadR-octanoyl-CoA complex crystallized in space group P65 22, with unit-cell parameters a = b = 59.7, c = 296.2 Å. Both crystal forms diffracted to 3.5 Å on a rotating-anode generator. In both crystal forms, the asymmetric unit contains one subunit. The protein is known to be a homodimer; each subunit consists of two domains of unknown fold. For the acyl-CoA-binding domain, a previously undetected sequence homology to PAS domains, in particular the photoactive yellow protein, is reported. [ABSTRACT FROM AUTHOR]

Growth rate and yield are fundamental features of microbial growth. However, we lack a mechanistic and quantitative understanding of the rate-yield relationship. Studies pairing computational predictions with experiments have shown the importance of maintenance energy and proteome allocation in explaining rate-yield tradeoffs and overflow metabolism. Recently, adaptive evolution experiments of Escherichia coli reveal a phenotypic diversity beyond what has been explained using simple models of growth rate versus yield. Here, we identify a two-dimensional rate-yield tradeoff in adapted E. coli strains where the dimensions are (A) a tradeoff between growth rate and yield and (B) a tradeoff between substrate (glucose) uptake rate and growth yield. We employ a multi-scale modeling approach, combining a previously reported coarse-grained small-scale proteome allocation model with a fine-grained genome-scale model of metabolism and gene expression (ME-model), to develop a quantitative description of the full rate-yield relationship for E. coli K-12 MG1655. The multi-scale analysis resolves the complexity of ME-model which hindered its practical use in proteome complexity analysis, and provides a mechanistic explanation of the two-dimensional tradeoff. Further, the analysis identifies modifications to the P/O ratio and the flux allocation between glycolysis and pentose phosphate pathway (PPP) as potential mechanisms that enable the tradeoff between glucose uptake rate and growth yield. Thus, the rate-yield tradeoffs that govern microbial adaptation to new environments are more complex than previously reported, and they can be understood in mechanistic detail using a multi-scale modeling approach. [ABSTRACT FROM AUTHOR]

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Cellular systems shift metabolic states by adjusting gene expression and enzyme activities to adapt to physiological and environmental changes. Biochemical and genetic studies are identifying how metabolic regulation affects the selection of metabolic phenotypes. However, how metabolism influences its regulatory architecture still remains unexplored. We present a new method of extreme pathway analysis (the minimal set of conically independent metabolic pathways) to deduce regulatory structures from pure pathway information. Applying our method to metabolic networks of human red blood cells and Escherichia coli, we shed light on how metabolic regulation are organized by showing which reactions within metabolic networks are more prone to transcriptional or allosteric regulation. Applied to a human genome-scale metabolic system, our method detects disease-associated reactions. Thus, our study deepens the understanding of the organizing principle of cellular metabolic regulation and may contribute to metabolic engineering, synthetic biology, and disease treatment. [ABSTRACT FROM AUTHOR]

Sepsis is a life threatening condition which produces multi-organ dysfunction with profound circulatory and cellular derangements. Administration of E.Coli endotoxin (LPS) produces systemic inflammatory effects of sepsis including disruption of endothelial barrier, and if severe enough death. Whole body periodic acceleration (pGz) is the headward-footward motion of the body. pGz has been shown to induce pulsatile shear stress to the endothelium, thereby releasing vascular and cardio protective mediators. The purpose of this study was to determine whether or not pGz performed as a pre-treatment or post-treatment strategy improves survival in a lethal murine endotoxin model.This study was designed as a prospective randomized controlled study in mice. pGz was performed in mice as pre-treatment (pGz-LPS, 3 days prior to LPS), post-treatment (LPS- pGz, 30 min after LPS) strategies or Control (LPS-CONT), in a lethal murine model of endotoxemia. Endotoxemia was induced with intraperitoneal injection of E.Coli LPS (40mg/kg). In a separate group of mice, a nonspecific nitric oxide synthase inhibitor (L-NAME) was provided in their drinking water and pGz-LPS and LPS-pGz performed to determine the effect of nitric oxide (NO) inhibition on survival. In another subset of mice, micro vascular leakage was determined. Behavioral scoring around the clock was performed in all mice at 30 min intervals after LPS administration, until 48 hrs. survival or death. LPS induced 100% mortality in LPS-CONT animals by 30 hrs. In contrast, survival to 48 hrs. occurred in 60% of pGz-LPS and 80% of LPS-pGz. L-NAME abolished the survival effects of pGz. Microvascular leakage was markedly reduced in both pre and post pGz treated animals and was associated with increased tyrosine kinase endothelial-enriched tunica interna endothelial cell kinase 2 (TIE2) receptor and its phosphorylation (p-TIE2). In a murine model of lethal endotoxemia, pGz performed as a pre or post treatment strategy significantly improved survival, and markedly reduced microvascular leakage. The effect was modulated, in part, by NO since a non-selective inhibitor of NO abolished the pGz survival effect. [ABSTRACT FROM AUTHOR]

The genetic regulatory network (GRN) plays a key role in controlling the response of the cell to changes in the environment. Although the structure of GRNs has been the subject of many studies, their large scale structure in the light of feedbacks from the metabolic network (MN) has received relatively little attention. Here we study the causal structure of the GRNs, namely the chain of influence of one component on the other, taking into account feedback from the MN. First we consider the GRNs of E. coli and B. subtilis without feedback from MN and illustrate their causal structure. Next we augment the GRNs with feedback from their respective MNs by including (a) links from genes coding for enzymes to metabolites produced or consumed in reactions catalyzed by those enzymes and (b) links from metabolites to genes coding for transcription factors whose transcriptional activity the metabolites alter by binding to them. We find that the inclusion of feedback from MN into GRN significantly affects its causal structure, in particular the number of levels and relative positions of nodes in the hierarchy, and the number and size of the strongly connected components (SCCs). We then study the functional significance of the SCCs. For this we identify condition specific feedbacks from the MN into the GRN by retaining only those enzymes that are essential for growth in specific environmental conditions simulated via the technique of flux balance analysis (FBA). We find that the SCCs of the GRN augmented by these feedbacks can be ascribed specific functional roles in the organism. Our algorithmic approach thus reveals relatively autonomous subsystems with specific functionality, or regulatory modules in the organism. This automated approach could be useful in identifying biologically relevant modules in other organisms for which network data is available, but whose biology is less well studied. [ABSTRACT FROM AUTHOR]

Hydrostatic pressure is an important physical stimulus which can cause various responses in bacterial cells. The survival and cellular processes of Escherichia coli under hydrostatic pressures between 10 MPa and 110 MPa have been studied. However, understanding bacterial responses to moderately elevated pressure of up to 10 MPa is useful for a range of different applications including for example in smart and responsive materials. In this study, the genetic responses of E. coli K-12 MG1655 to 1 MPa pressure was examined using transcriptomic analysis by RNA-Seq. The results show that 101 genes were differentially expressed under 1 MPa pressure in E. coli cells, with 85 of them up-regulated. The analysis suggested that some genes were over expressed to adapt the increase of oxygen levels in our system, and several functional categories are involved including oxidative stress responses, Fe-S cluster assembly and iron acquisition. Two differentially expressed genes azuC and entC were further investigated using RT-qPCR, and GFP reported strains of those two genes were created, AG1319 (PazuC azuC-msfgfp) and AG1321 (PentC entC-msfgfp). A linear response of azuC expression was observed between 0 MPa to 1 MPa by monitoring the fluorescence signal of strain AG1319 (PazuC azuC-msfgfp). This study is the first report to demonstrate the genetic response of bacterial cells under 1 MPa hydrostatic pressure, and provides preliminary data for creating pressure sensing bacterial strains for a wide range of applications. [ABSTRACT FROM AUTHOR]

Genome-scale models of metabolism and macromolecular expression (ME-models) explicitly compute the optimal proteome composition of a growing cell. ME-models expand upon the well-established genome-scale models of metabolism (M-models), and they enable a new fundamental understanding of cellular growth. ME-models have increased predictive capabilities and accuracy due to their inclusion of the biosynthetic costs for the machinery of life, but they come with a significant increase in model size and complexity. This challenge results in models which are both difficult to compute and challenging to understand conceptually. As a result, ME-models exist for only two organisms (Escherichia coli and Thermotoga maritima) and are still used by relatively few researchers. To address these challenges, we have developed a new software framework called COBRAme for building and simulating ME-models. It is coded in Python and built on COBRApy, a popular platform for using M-models. COBRAme streamlines computation and analysis of ME-models. It provides tools to simplify constructing and editing ME-models to enable ME-model reconstructions for new organisms. We used COBRAme to reconstruct a condensed E. coli ME-model called iJL1678b-ME. This reformulated model gives functionally identical solutions to previous E. coli ME-models while using 1/5 the number of free variables and solving in less than 10 minutes, a marked improvement over the 6 hour solve time of previous ME-model formulations. Errors in previous ME-models were also corrected leading to 52 additional genes that must be expressed in iJL1678b-ME to grow aerobically in glucose minimal in silico media. This manuscript outlines the architecture of COBRAme and demonstrates how ME-models can be created, modified, and shared most efficiently using the new software framework. [ABSTRACT FROM AUTHOR]

A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0–10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing. [ABSTRACT FROM AUTHOR]

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads. [ABSTRACT FROM AUTHOR]

Previous screening of a single-gene knockout library consisting of 3,908 disrupted-mutant strains allowed us to identify 51 thermotolerant genes that are essential for survival at a critical high temperature (CHT) in Escherichia coli [Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M (2011) PLoS ONE 6: e20063]. In this study, we identified another 21 thermotolerant genes. E. coli thus has 72 thermotolerant genes in total. The genes are classified into 8 groups: genes for energy metabolism, outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control, cell division and transporters. This classification and physiological analysis indicate the existence of fundamental strategies for survival at a CHT, which seems to exclude most of the heat shock responses. [ABSTRACT FROM AUTHOR]

The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and in vitro biochemical assays. Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur. [ABSTRACT FROM AUTHOR]

We experimentally and numerically investigate the evolutionary dynamics of four competing strains of E. coli with differing expansion velocities in radially expanding colonies. We compare experimental measurements of the average fraction, correlation functions between strains, and the relative rates of genetic domain wall annihilations and coalescences to simulations modeling the population as a one-dimensional ring of annihilating and coalescing random walkers with deterministic biases due to selection. The simulations reveal that the evolutionary dynamics can be collapsed onto master curves governed by three essential parameters: (1) an expansion length beyond which selection dominates over genetic drift; (2) a characteristic angular correlation describing the size of genetic domains; and (3) a dimensionless constant quantifying the interplay between a colony’s curvature at the frontier and its selection length scale. We measure these parameters with a new technique that precisely measures small selective differences between spatially competing strains and show that our simulations accurately predict the dynamics without additional fitting. Our results suggest that the random walk model can act as a useful predictive tool for describing the evolutionary dynamics of range expansions composed of an arbitrary number of genotypes with different fitnesses. [ABSTRACT FROM AUTHOR]

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities. [ABSTRACT FROM AUTHOR]

The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

Biomimetic synthesis of metal nanoparticles (NPs) is safe and eco‐friendly; therefore, find diverse applications. Considering this, the soil fungi Penicillium chrysogenum strain Fungal germplasm collection centre/ BLS1 was isolated, characterized and explored to synthesize extracellular silver NPs (AgNPs) under optimised conditions. The synthesis of AgNPs was investigated using ultraviolet (UV)–visible spectroscopy, Fourier‐transform infra‐red spectroscopy (FTIR), transmission electron microscope (TEM) and dynamic light scattering (DLS) analysis. Process optimisation exhibited AgNPs synthesis within 8 h using 2 mM AgNO3 at pH 11 and temperature 70°C. TEM analysis revealed polydispersed ellipsoidal shaped AgNPs with average particle size 96.8 nm as measured by DLS. AgNPs showed negative zeta potential that confers surface stability in solution. FTIR spectra confirmed the presence of protein bound to AgNPs. Antibacterial activity against Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus by the AgNPs (100 ppm) was demonstrated by counting colony forming unit, disc diffusion, and growth kinetics assay. Additionally radial assay revealed antifungal activity of AgNPs (100 ppm) against phytopathogenic fungi Sclerotinia sclerotiorum Microbial type culture collection 8785. Furthermore, AgNPs (100 ppm) did not show any cytotoxic effects on human Red blood cells. Therefore, this novel fungal strain can be utilised for biofabrication of AgNPs under optimised conditions and have shown strong antimicrobial property. [ABSTRACT FROM AUTHOR]

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators. [ABSTRACT FROM AUTHOR]

In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases. [ABSTRACT FROM AUTHOR]

In phenotypically heterogeneous microbial populations, the decision to adopt one or another phenotype is often stochastically regulated. However, how this stochasticity affects interactions between competing microbes in mixed communities is difficult to assess. One example of such an interaction system is the competition of an Escherichia coli strain C, which performs division of labor between reproducers and self-sacrificing toxin producers, with a toxin-sensitive strain S. The decision between reproduction or toxin production within a single C cell is inherently stochastic. Here, combining experimental and theoretical approaches, we demonstrate that this stochasticity in the initial phase of colony formation is the crucial determinant for the competition outcome. In the initial phase (t < 12h), stochasticity influences the formation of viable C clusters at the colony edge. In the subsequent phase, the effective fitness differences (set primarily by the degree of division of labor in the C strain population) dictate the deterministic population dynamics and consequently competition outcome. In particular, we observe that competitive success of the C strain is only found if (i) a C edge cluster has formed at the end of the initial competition phase and (ii) the beneficial and detrimental effects of toxin production are balanced, which is the case at intermediate toxin producer fractions. Our findings highlight the importance of stochastic processes during the initial phase of colony formation, which might be highly relevant for other microbial community interactions in which the random choice between phenotypes can have long-lasting consequences for community fate. [ABSTRACT FROM AUTHOR]

Human α and β-defensins are cationic antimicrobial peptides characterized by three disulfide bonds with a triple stranded β-sheet motif. It is presumed that interaction with the bacterial cell surface and membrane permeabilization by defensins is an important step in the killing process. In this study, we have compared interactions of three human α-defensins HNP3, HNP4, HD5 and human β-defensins HBD1-4 that are active against Escherichia coli, with its cell surface and inner membrane as well as negatively charged model membranes. We have also included the inactive α-defensin HD6 in the study. Among the α-defensins, HNP4, HD5 and HD6 were more effective in increasing the zeta potential as compared to HNP3. Among the β-defensins, HBD1 was the least effective in increasing the zeta potential. The zeta potential modulation data indicate variations in the surface charge neutralizing ability of α- and β-defensins. Comparison of E. coli inner membrane and model membrane permeabilizing abilities indicated that HD5, HD6 and HBD1 do not permeabilize membranes. Although HBD4 does not permeabilize model membranes, considerable damage to the inner membrane of E. coli is observed. Our data indicate that mammalian defensins do not kill E. coli by a simple mechanism involving membrane permeabilization though their antibacterial potencies are very similar. [ABSTRACT FROM AUTHOR]

A stochastic version of the Barkai-Leibler model of chemotaxis receptors in Escherichia coli is studied here with the goal of elucidating the effects of intrinsic network noise in their conformational dynamics. The model was originally proposed to explain the robust and near-perfect adaptation of E. coli observed across a wide range of spatially uniform attractant/repellent (ligand) concentrations. In the model, a receptor is either active or inactive and can stochastically switch between the two states. The enzyme CheR methylates inactive receptors while CheB demethylates active receptors and the probability for a receptor to be active depends on its level of methylation and ligand occupation. In a simple version of the model with two methylation sites per receptor (M = 2), we show rigorously, under a quasi-steady state approximation, that the mean active fraction of receptors is an ultrasensitive function of [CheR]/[CheB] in the limit of saturating receptor concentration. Hence the model shows zero-order ultrasensitivity (ZOU), similar to the classical two-state model of covalent modification studied by Goldbeter and Koshland (GK). We also find that in the limits of extremely small and extremely large ligand concentrations, the system reduces to two different two-state GK modules. A quantitative measure of the spontaneous fluctuations in activity is provided by the variance in the active fraction, which is estimated mathematically under linear noise approximation (LNA). It is found that peaks near the ZOU transition. The variance is a non-monotonic, but weak function of ligand concentration and a decreasing function of receptor concentration. Gillespie simulations are also performed in models with M = 2, 3 and 4. For M = 2, simulations show excellent agreement with analytical results obtained under LNA. Numerical results for M = 3 and M = 4 are qualitatively similar to our mathematical results in M = 2; while all the models show ZOU in mean activity, the variance is found to be smaller for larger M. The magnitude of receptor noise deduced from available experimental data is consistent with our predictions. A simple analysis of the downstream signaling pathway shows that this noise is large enough to affect the motility of the organism, and may have a beneficial effect on it. The response of mean receptor activity to small time-dependent changes in the external ligand concentration is computed within linear response theory, and found to have a bilobe form, in agreement with earlier experimental observations. [ABSTRACT FROM AUTHOR]

Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential. [ABSTRACT FROM AUTHOR]

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo. [ABSTRACT FROM AUTHOR]

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended ‘lid’ region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries. [ABSTRACT FROM AUTHOR]