Mainly on the Plane: Deep Subsurface Bacterial Proteins Bind and Alter Clathrate Structure

ABSTRACTGas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analog for structure II gas clathrate. When grown in the presence of CbpAs, THF clathrate was polycrystalline and plate-like instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a plate-like structure during gas clathrate transportation.Table of Contents Graphic

Prebiotics for Lactose Intolerance: Variability in Galacto-Oligosaccharide Utilization by Intestinal Lactobacillus rhamnosus

Lactose intolerance, characterized by a decrease in host lactase expression, affects approximately 75% of the world population. Galacto-oligosaccharides (GOS) are prebiotics that have been shown to alleviate symptoms of lactose intolerance and to modulate the intestinal microbiota, promoting the growth of beneficial microorganisms. We hypothesized that mechanisms of GOS utilization by intestinal bacteria are variable, impacting efficacy and response, with differences occurring at the strain level. This study aimed to determine the mechanisms by which human-derived Lactobacillus rhamnosus strains metabolize GOS. Genomic comparisons between strains revealed differences in carbohydrate utilization components, including transporters, enzymes for degradation, and transcriptional regulation, despite a high overall sequence identity (>95%) between strains. Physiological and transcriptomics analyses showed distinct differences in carbohydrate metabolism profiles and GOS utilization between strains. A putative operon responsible for GOS utilization was identified and characterized by genetic disruption of the 6-phospho-β-galactosidase, which had a critical role in GOS utilization. Our findings highlight the importance of strain-specific bacterial metabolism in the selection of probiotics and synbiotics to alleviate symptoms of gastrointestinal disorders including lactose intolerance.

Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.

The plastid genome of some eustigmatophyte algae harbours a bacteria-derived six-gene cluster for biosynthesis of a novel secondary metabolite

Acquisition of genes by plastid genomes (plastomes) via horizontal gene transfer (HGT) seems to be a rare phenomenon. Here, we report an interesting case of HGT revealed by sequencing the plastomes of the eustigmatophyte algae Monodopsis sp. MarTras21 and Vischeria sp. CAUP Q 202. These plastomes proved to harbour a unique cluster of six genes, most probably acquired from a bacterium of the phylum Bacteroidetes, with homologues in various bacteria, typically organized in a conserved uncharacterized putative operon. Sequence analyses of the six proteins encoded by the operon yielded the following annotation for them: (i) a novel family without discernible homologues; (ii) a new family within the superfamily of metallo-dependent hydrolases; (iii) a novel subgroup of the UbiA superfamily of prenyl transferases; (iv) a new clade within the sugar phosphate cyclase superfamily; (v) a new family within the xylose isomerase-like superfamily; and (vi) a hydrolase for a phosphate moiety-containing substrate. We suggest that the operon encodes enzymes of a pathway synthesizing an isoprenoid–cyclitol-derived compound, possibly an antimicrobial or other protective substance. To the best of our knowledge, this is the first report of an expansion of the metabolic capacity of a plastid mediated by HGT into the plastid genome.

Identification of a Putative Operon Involved in Fructooligosaccharide Utilization by Lactobacillus paracasei

ABSTRACTThe growth and activity of someLactobacillusandBifidobacteriumstrains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments ofLactobacillus paracasei1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding β-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated thefosoperon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putativefosoperon was determined and consists offosABCDXEgenes, which encode a putative fructose/mannose PTS (FosABCDX) and a β-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of thefosEgene led to impaired growth on FOS and other β-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested thatfosABCDXEwas cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

ABSTRACT This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3′-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.

Role of Protein Kinase G in Growth and Glutamine Metabolism of Mycobacterium bovis BCG

ABSTRACT The survival of pathogenic mycobacteria in macrophages requires the eukaryotic enzyme-like serine/threonine protein kinase G. This kinase with unknown specificity is secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake. Here, we report that the deletion of pknG did not affect either glutamine uptake or intracellular glutamine concentrations. In vitro growth of Mycobacterium bovis BCG lacking pknG was identical to that of the wild type. We conclude that in M. bovis BCG, glutamine metabolism is not regulated by protein kinase G.