Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.192, #3)

An overview of in silico protein function prediction by Roy D. Sleator; Paul Walsh (pp. 151-155).

As the protein databases continue to expand at an exponential rate, fed by daily uploads from multiple large scale genomic and metagenomic projects, the problem of assigning a function to each new protein has become the focus of significant research interest in recent times. Herein, we review the most recent advances in the field of automated function prediction (AFP). We begin by defining what is meant by biological “function” and the means of describing such functions using standardised machine readable ontologies. We then focus on the various function-prediction programs available, both sequence and structure based, and outline their associated strengths and weaknesses. Finally, we conclude with a brief overview of the future challenges and outstanding questions in the field, which still remain unanswered.

Keywords: Protein function; Homology-based transfer; Ontologies; Sequence and structure Motifs

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant by Hermansyah; Walter A. Laviña; Minetaka Sugiyama; Yoshinobu Kaneko; Satoshi Harashima (pp. 157-165).

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.

Keywords: PTP2 ; MSG5 ; Protein phosphatase; Protein Kinase; Calcium sensitive; Saccharomyces cerevisiae

Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri by Liu Liu; Mahesh Dharne; Porteen Kannan; Allen Smith; Jianghong Meng; Mingtao Fan; Tara L. Boren; Ryan T. Ranallo; Arvind A. Bhagwat (pp. 167-174).

Osmoregulated periplasmic glucans (OPGs) of food- and water-borne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100% glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominant backbone polymer chain length was seven glucose residues. Individual genes from the opg gene family comprising of a bicistronic operon opgGH, opgB, opgC and opgD were mutagenized to study their effect on OPGs synthesis, growth in hypo-osmotic media and ability to invade HeLa cells. Mutation in opgG and opgH abolished OPGs biosynthesis, and mutants experienced longer lag time to initiate growth in hypo-osmotic media. Longer lag times to initiate growth in hypo-osmotic media were also observed for opgC and opgD mutants but not for opgB mutant. All opg mutants were able to infect HeLa cells, and abolition of OPGs synthesis did not affect actin polymerization or plaque formation. Ability to synthesize OPGs was beneficial to bacteria in order to initiate growth under low osmolarity conditions, in vitro mammalian cell invasion assays, however, could not discriminate whether OPGs were required for basic aspect of Shigella virulence.

Keywords: Periplasmic glucans; Low osmolarity; Food- and water-borne Shigellosis

Molecular diversity of katG genes in the soil bacteria Comamonas by Jana Godočíková; Marcel Zámocký; Mária Bučková; Christian Obinger; Bystrík Polek (pp. 175-184).

Three complete katG genes coding for bifunctional catalase-peroxidases (KatGs) from the β-proteobacterium Comamonas terrigena and two related strains of Comamonas testosteroni have been cloned and sequenced. Catalase-peroxidases are unique bifunctional enzymes known to be expressed in these soil bacteria in response to environmental and/or oxidative stress. The evolutionary and structural diversity of these enzymes is investigated based on multiple sequence alignment and comprehensive phylogenetic analysis. The reconstructed phylogenetic tree and well-known structure–function relationships were applied to inspect the conservation of essential residues. Observed diversity is discussed with respect to the fact that KatGs are distinctive gene-duplicated peroxidases comprising a N-terminal (enzymatically active) and a C-terminal (heme-less) domain. The unique promoter motifs regulating katG transcription in four strains of Comamonas were detected and compared with E. coli katG promoter. The relationship between the promoter sequences and the corresponding expression levels was analyzed. A significant difference in heat shock-inducible catalatic and peroxidatic activities between E. coli K12 and Comamonas terrigena & testosteroni strains was observed. The peculiar variability in gene-coding sequences appears to be more significant for such activity output among Comamonas strains than differences in their promoter regions. The functional role of observed increased diversity in the C-terminal domain is discussed with respect to potential modification of catalytic features at the N-terminal domain that could be relevant for these soil bacteria to cope with stressors.

Keywords: Catalase-peroxidase; KatG gene; Comamonas terrigena ; Comamonas testosteroni ; Molecular diversity; Positive darwinian selection

Environmental Escherichia coli occur as natural plant growth-promoting soil bacterium by Chandra Shekhar Nautiyal; Ateequr Rehman; Puneet Singh Chauhan (pp. 185-193).

Currently, it is presumed that Escherichia coli is not a normal inhabitant of the soil. Soilborne E. coli strains were isolated from broad range of 7 geoclimatic zones of India, indicating that E. coli can survive and thrive under different extreme soil conditions. Diversity among E. coli strains from widely separated geographic regions using enterobacterial repetitive intergenic consensus (ERIC)-PCR did not reveal any relationships between the genotypes and the source of isolation. Inoculation of maize (Zea mays cv. Arkil) seeds with E. coli NBRIAR3 (NBRIAR3) significantly enhanced (P < 0.05) plant growth and nutrient uptake, when compared with uninoculated control. Presence or absence of NBRIAR3 did not affect significantly (P < 0.05) diversity indexes, using substrate utilization patterns on the Biolog Eco plates. Clone libraries based on 16S rRNA gene from rhizosphere of maize plants demonstrated rather similar phylotype diversity from the uninoculated control and NBRIAR3-treated rhizosphere soil, which further indicated that NBRIAR3 did not exert a major influence on the overall bacterial diversity. The methodological approach described in this study supports the idea that E. coli should be treated as native soil bacterium instead of as an “indicator” of the possible presence of other fecal coliform bacteria.

Keywords: Escherichia coli ; Fecal coliforms; Environment; Native soil bacteria; Plant growth promotion; Rhizosphere

Subunit–subunit interactions are weakened in mutant forms of acetohydroxy acid synthase insensitive to valine inhibition by Martina Kyselková; Jiří Janata; Markéta Ságová-Marečková; Jan Kopecký (pp. 195-200).

In acetohydroxy acid synthase from Streptomyces cinnamonensis mutants affected in valine regulation, the impact of mutations on interactions between the catalytic and the regulatory subunits was examined using yeast two-hybrid system. Mutations in the catalytic and the regulatory subunits were projected into homology models of the respective proteins. Two changes in the catalytic subunit, E139A (α domain) and ΔQ217 (β domain), both located on the surface of the catalytic subunit dimer, lowered the interaction with the regulatory subunit. Three consecutive changes in the N-terminal part of the regulatory subunit were examined. Changes G16D and V17D in a loop and adjacent α-helix of ACT domain affected the interaction considerably, indicating that this region might be in contact with the catalytic subunit during allosteric regulation. In contrast, the adjacent mutation L18F did not influence the interaction at all. Thus, L18 might participate in valine binding or conformational change transfer within the regulatory subunits. Shortening of the regulatory subunit to 107 residues reduced the interaction essentially, suggesting that the C-terminal part of the regulatory subunit is also important for the catalytic subunit binding.

Keywords: Streptomyces cinnamonensis ; Acetohydroxy acid synthase; Subunit–subunit interaction; Yeast two-hybrid system; Allosteric regulation; ACT domain

Isoeugenol monooxygenase and its putative regulatory gene are located in the eugenol metabolic gene cluster in Pseudomonas nitroreducens Jin1 by Ji-Young Ryu; Jiyoung Seo; Tatsuya Unno; Joong-Hoon Ahn; Tao Yan; Michael J. Sadowsky; Hor-Gil Hur (pp. 201-209).

The plant-derived phenylpropanoids eugenol and isoeugenol have been proposed as useful precursors for the production of natural vanillin. Genes involved in the metabolism of eugenol and isoeugenol were clustered in region of about a 30 kb of Pseudomonas nitroreducens Jin1. Two of the 23 ORFs in this region, ORFs 26 (iemR) and 27 (iem), were predicted to be involved in the conversion of isoeugenol to vanillin. The deduced amino acid sequence of isoeugenol monooxygenase (Iem) of strain Jin1 had 81.4% identity to isoeugenol monooxygenase from Pseudomonas putida IE27, which also transforms isoeugenol to vanillin. Iem was expressed in E. coli BL21(DE3) and was found to lead to isoeugenol to vanillin transformation. Deletion and cloning analyses indicated that the gene iemR, located upstream of iem, is required for expression of iem in the presence of isoeugenol, suggesting it to be the iem regulatory gene. Reverse transcription, real-time PCR analyses indicated that the genes involved in the metabolism of eugenol and isoeugenol were differently induced by isoeugenol, eugenol, and vanillin.

Keywords: Isoeugenol monooxygenase; Pseudomonas ; Vanillin; Eugenol; Regulator

Control of secondary metabolism by farX, which is involved in the γ-butyrolactone biosynthesis of Streptomyces lavendulae FRI-5 by Shigeru Kitani; Masashi Doi; Tomohito Shimizu; Asa Maeda; Takuya Nihira (pp. 211-220).

The γ-butyrolactone signaling system is distributed widely among streptomycetes as an important regulatory mechanism of antibiotic production and/or morphological differentiation. IM-2 [(2R,3R,1′R)-2-(1′-hydroxybutyl)-3-hydroxymethyl-γ-butanolide] is a γ-butyrolactone that switches off the production of d-cycloserine but switches on the production of several nucleoside antibiotics as well as blue pigment in Streptomyces lavendulae FRI-5. farX is a member of the afsA-family genes, which are proposed to encode enzymes involved in γ-butyrolactone biosynthesis. Disruption of farX caused overproduction of d-cycloserine, and abolished production of nucleoside antibiotic and blue pigment with the loss of IM-2 production. The finding that all phenotypic changes observed in the farX disruptant were restored by the addition of exogenous IM-2 suggested that FarX plays a biosynthetic role in IM-2 production. Transcriptional comparison between the wild-type strain and the farX disruptant revealed that, in addition to already known genes farR1 and farR2, several other genes (farR4, farD, and farE) are under the transcriptional regulation of IM-2. Furthermore, the fact that farX transcription is under the control of IM-2 suggested that S. lavendulae FRI-5 has a fine-tuning system to control γ-butyrolactone production.

Keywords: Signal transduction; d-cycloserine; Diffusing signaling molecule; Negative autoregulatory circuit

Effect of vitamin A treatment on superoxide dismutase-deficient yeast strains by Rafael Roehrs; Daniela R. J. Freitas; Aoi Masuda; João A. P. Henriques; Temenouga N. Guecheva; Ana-Ligia L. P. Ramos; Jenifer Saffi (pp. 221-228).

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Δ) and CuZnSOD/MnSOD (sod1Δsod2Δ). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.

Keywords: Vitamin A; Reactive oxygen species (ROS); Saccharomyces cerevisiae ; Oxidative stress; Paraquat

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes by Jie Liu; En Tao Wang; Da Wei Ren; Wen Xin Chen (pp. 229-234).

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.

Keywords: Nodulation; Agrobacterium ; Sinorhizobium ; Endophyte; Woody legume; Nodule morphology

L-forms and the unusual formation of progeny in cell wall-less bacteria: recognizing the old roots of new science by Hans Herbert Martin (pp. 235-236).

Sections

Personal tools

Archives of Microbiology (v.192, #3)

Sections

Personal tools

Local resources

Archives of Microbiology (v.192, #3)