Identification, Characterization, and Hydrolase Producing Performance of Thermophilic Bacteria: Geothermal Hot Springs in Eastern and Southeastern Anatolia Region of Turkey

In the last twenty years, researchers have increasingly focused on the rich microorganism-based diversity of natural hot spring resources to explore the benefits of thermophiles in industrial and biotechnological fields. For this purpose, a total of 83 thermophilic bacilli were isolated from 7 different geothermal hot springs (at temperatures ranging between 40 and 85 ◦ C) located in the east and Southeastern of Turkey. These isolates were identified by different methods such as physiological, morphological, biochemical, molecular properties. According to 16S rRNA gene sequence analysis, 5 different isolates ( Bacillus coagulans , Bacillus licheniformis , Bacillus subtilis , Bacillus thuringiensis , and Geobacillus kaustophilus ) were identified. It was observed that B. licheniformis and B. subtilis were the most species obtained from the researched hot spring sources. Phylogenetic relationships of isolates were evaluated with the help of a phylogenetic tree. The conditions of bacterial isolates to synthesize various hydrolytic enzymes such as protease, cellulase, lipase, and amylase were investigated. When the potential of isolates to produce hydrolytic enzymes was examined, protease 73 (88%), cellulase 34 (41%), lipase 69 (83%), and amylase 68 (82%) were detected. All isolates have at least one or more extracellular protease, cellulase, amylase, or lipase activity. Besides, 32.8% (27) of bacterial isolates were able to synthesize all of the hydrolytic enzymes.

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4–9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition.

Selección e identificación de una nueva bacteria productora de pectinasa a partir de fuentes geotermales

Los microorganismos son una fuente potencial de enzimas. Las bacterias termófilas producen enzimas termoestables, como las pectinasas, que permiten obtener una serie de productos que hidrolizan pectinas, heteropolisacáridos, componentes principales de la capa media de la pared celular de la piel de los cítricos (naranjas, limones y mandarinas), manzanas, melocotones y otros vegetales. El cultivo bacteriano productor de pectinasas se seleccionó de bacterias aisladas de géiseres de aguas termales. Las muestras se sembraron en medio líquido y sólido, se incubaron a 60 °C durante 24 a 48 horas respectivamente, observándose halos de hidrólisis como indicativos de producción de pectinasa. Las bacterias que formaron los halos se sembraron en medio de producción de pectinasas, evaluando su actividad enzimática y producción de proteínas. Se aislaron veinte cultivos bacterianos gram positivos, de los cuales 12 fueron productores de pectinasas, siendo el cultivo LBE-P4 el de mayor actividad enzimática con 0.154 U/ml y 0.069 mg/ml de proteínas totales, que fue identificado molecularmente como Geobacillus kaustophilus LBE-P4.

Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis . The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus . When iolQ was inactivated in G. kaustophilus , not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

Identification of a Repressor for the Two iol Operons Required for Inositol Catabolism in Geobacillus Kaustophilus

BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster consists of two tandem operons induced in the presence of inositol; however, the mechanism underlying the induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding a scyllo-inositol dehydrogenase, and its homolog in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and termed as iolQ in G. kaustophilus.ResultsWhen iolQ was inactivated, not only the myo-inositol dehydrogenase activity in the cell due to the expression of gk1899 but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal His-tag fusion in Escherichia coli and subjected to the in vitro gel mobility shift assay to examine its DNA binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions, and its DNA binding was antagonized by myo-inositol. Moreover, DNase I footprint analyses were conducted to determine the two binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to be a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R = A or G, W = A or T, S = G or C, and Y = C or T).ConclusionIolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.