Distribution of Cepacian Biosynthesis Genes among Environmental and Clinical Burkholderia Strains and Role of Cepacian Exopolysaccharide in Resistance to Stress Conditions

ABSTRACT The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a B urkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

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Ferrous Iron- and Sulfur-Induced Genes in Sulfolobus metallicus

Applied and Environmental Microbiology ◽

10.1128/aem.02589-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2491-2497 ◽

Cited By ~ 63

Author(s):

Stephan Bathe ◽

Paul R. Norris

Keyword(s):

Reverse Transcription ◽

Ferrous Iron ◽

Subtractive Hybridization ◽

Open Reading Frames ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Induced Genes ◽

Reading Frames ◽

Sulfur Oxygenase Reductase

ABSTRACT Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

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Global Gene Expression Profile for Swarming Bacillus cereus Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00245-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5149-5156 ◽

Cited By ~ 28

Author(s):

Sara Salvetti ◽

Karoline Faegri ◽

Emilia Ghelardi ◽

Anne-Brit Kolstø ◽

Sonia Senesi

Keyword(s):

Bacillus Cereus ◽

Transcriptional Response ◽

Global Gene Expression ◽

Solid Surfaces ◽

Content Type ◽

Reverse Transcription Pcr ◽

Genome Wide ◽

Genes Encoding ◽

Hemolysin Bl

ABSTRACTBacillus cereuscan use swarming to move over and colonize solid surfaces in different environments. This kind of motility is a collective behavior accompanied by the production of long and hyperflagellate swarm cells. In this study, the genome-wide transcriptional response ofB. cereusATCC 14579 during swarming was analyzed. Swarming was shown to trigger the differential expression (>2-fold change) of 118 genes. Downregulated genes included those required for basic cellular metabolism. In accordance with the hyperflagellate phenotype of the swarm cell, genes encoding flagellin were overexpressed. Some genes associated with K+transport, phBC6A51 phage genes, and the binding component of the enterotoxin hemolysin BL (HBL) were also induced. Quantitative reverse transcription-PCR (qRT-PCR) experiments indicated an almost 2-fold upregulation of the entirehbloperon during swarming. Finally, BC1435 and BC1436, orthologs ofliaI-liaHthat are known to be involved in the resistance ofBacillus subtilisto daptomycin, were upregulated under swarming conditions. Accordingly, phenotypic assays showed reduced susceptibility of swarmingB. cereuscells to daptomycin, and Pspac-induced hyper-expression of these genes in liquid medium highlighted the role of BC1435 and BC1436 in the response ofB. cereusto daptomycin.

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Serine/Threonine Protein Kinase Stk Is Required for Virulence, Stress Response, and Penicillin Tolerance in Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.05360-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4201-4209 ◽

Cited By ~ 32

Author(s):

Julia Bugrysheva ◽

Barbara J. Froehlich ◽

Jeffrey A. Freiberg ◽

June R. Scott

Keyword(s):

Streptococcus Pyogenes ◽

Fatty Acid Biosynthesis ◽

Group A Streptococcus ◽

Acid Biosynthesis ◽

Regulation Of Expression ◽

Content Type ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Group A

ABSTRACTGenes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogenStreptococcus pyogenes(group A streptococcus [GAS]). We report that in GAS,stkis required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that thestkdeletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.

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Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Microbiology ◽

10.1099/mic.0.074286-0 ◽

2014 ◽

Vol 160 (3) ◽

pp. 623-634 ◽

Cited By ~ 7

Author(s):

Tetsu Shimizu ◽

Akira Nakamura

Keyword(s):

Constitutive Expression ◽

Transcriptional Regulator ◽

Pcr Analysis ◽

Mobility Shift ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Dnase I Footprinting ◽

Catabolic Genes ◽

Genes Encoding ◽

Mobility Shift Assay

Five genes encoding enzymes required for l-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of l-gluconate or d-idonate, a catabolite of l-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. d-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the l-gluconate catabolic pathway and d-galactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

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Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

International Journal of Molecular Sciences ◽

10.3390/ijms23020772 ◽

2022 ◽

Vol 23 (2) ◽

pp. 772

Author(s):

Rosaura Rodicio ◽

Hans-Peter Schmitz ◽

Jürgen J. Heinisch

Keyword(s):

Kluyveromyces Lactis ◽

Regulation Of Gene Expression ◽

Carbon Sources ◽

Pentose Phosphate ◽

Gene Encoding ◽

Physiological Characterization ◽

Genes Encoding ◽

Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

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Redox-active secondary metabolites act as interspecies modulators of antibiotic resilience

10.1101/2021.12.01.470848 ◽

2021 ◽

Author(s):

Lucas A. Meirelles ◽

Dianne K. Newman

Keyword(s):

Secondary Metabolites ◽

Burkholderia Cepacia ◽

Defense Responses ◽

Microbial Interactions ◽

Opportunistic Pathogens ◽

Burkholderia Gladioli ◽

Redox Active ◽

Wide Range ◽

Genes Encoding

ABSTRACTBacterial opportunistic pathogens make a wide range of secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of two redox-active secondary metabolites, pyocyanin and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin is made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. Both molecules alter the susceptibility profile of pathogenic species within the “Burkholderia cepacia complex” to different antibiotics, either antagonizing or potentiating their effects, depending on the drug’s class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

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Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats’ Rumen by Metagenomic Deep Sequencing

Animals ◽

10.3390/ani11113257 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3257

Author(s):

Trong-Khoa Dao ◽

Thi-Huyen Do ◽

Ngoc-Giang Le ◽

Hong-Duong Nguyen ◽

Thi-Quy Nguyen ◽

...

Keyword(s):

Deep Sequencing ◽

Gene Encoding ◽

Amylolytic Enzymes ◽

E Coli ◽

Lignocellulose Conversion ◽

Genes Encoding ◽

Abundant Genus ◽

First Time ◽

Goat Rumen

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats’ rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5–6.5, temperature 40–50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

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Phenotypic Plasticity for Growth and Nutrient Uptake in Milk Thistle under Salt Stress: Modulatory Role of Soil Supplementations with Plant Growth Promoters

International Journal of Agriculture and Biology ◽

10.17957/ijab/15.1718 ◽

2021 ◽

Vol 25 (03) ◽

pp. 692-700

Author(s):

Noreen Zahra

Keyword(s):

Salt Stress ◽

Phenotypic Plasticity ◽

Plant Growth ◽

Salinity Stress ◽

Arid Zone ◽

Milk Thistle ◽

Growth Promoters ◽

Plant Growth Promoters ◽

Semi Arid

Salinity stress negatively affects key physiological phenomena in plants while plants show great variability and respond differentially for tolerance to salt stress. Usually, nutrients imbalances affect specific plant tissues and physiological processes which are requisite for normal plant growth and development. The aim of this two-year (2017 and 2018) simulated field study was to investigate phenotypic plasticity for growth, relative leaf water content (RLWC) and nutrient status in milk thistle [Silybum marianum (L.) Gaertn.] ecotypes and the potential role of soil supplementation with pre-optimized levels of plant growth promoters (PGPs) in modulating these attributes under control and salinity (12 dS/m) stress. Four ecotypes of milk thistle were collected from three ecologically distinct zones including Faisalabad (FSD) and Kalar Kahar (KK) – semi-arid zone, Gujranwala (GUJ) – hot semi-arid zone and Quetta (QTA) – cool semi-arid zone. The studied nutrients were nitrate-N, phosphate-P, sulfate-S, sodium (Na), potassium (K) and calcium (Ca). The soil supplemented PGPs, applied with irrigation water, were ascorbic acid (AsA), thiourea (TU) and moringa leaf extract (MLE) at 250 μM, 500 μM and 3%, respectively of soil moisture content at field capacity. Results indicated that soil supplementation of PGPs in the field conditions is a feasible approach for enhancing nutrient uptake of milk thistle ecotypes under salt stress, while the effect of salinity stress restricted the uptake of the studied nutrients and caused their imbalance. Although the salinity stress reduced shoot and root dry matter, RLWC and restricted the uptake of these nutrients irrespective of ecotypes, the levels of nitrate-N, phosphate-P, K, sulfate-S, Ca, and RWC contents increased more with the soil supplementation of AsA followed by MLE as compared to other soil supplements in both the study years. Among the ecotypes, QTA followed by KK and FSD ecotypes gained more dry weight with greater leaf RWC and higher tissue nutrient contents due to PGPs under salt stress. The principal component analysis and correlation data revealed the existence of distinct phenotypic plasticity in the milk thistle ecotypes for nutrient acquisition with soil supplementation of PGPs under salinity stress. To conclude, ecotypes from QTA and KK were more promising than the others while AsA and MLE were better soil supplements in improving shoot and root nutrients under salt stress. © 2021 Friends Science Publishers

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The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein

International Journal of Molecular Sciences ◽

10.3390/ijms222313109 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13109

Author(s):

Carmen-Lisset Flores ◽

Joaquín Ariño ◽

Carlos Gancedo

Keyword(s):

Yarrowia Lipolytica ◽

Kinase Activity ◽

Rna Seq ◽

Wild Type ◽

Gene Encoding ◽

Moonlighting Protein ◽

Genes Expression ◽

Genes Encoding ◽

Utilization Pathway

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

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Insights into the Function and Horizontal Transfer of Isoproturon Degradation Genes (pdmAB) in a Biobed System

Applied and Environmental Microbiology ◽

10.1128/aem.00474-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Veronika Storck ◽

Sara Gallego ◽

Sotirios Vasileiadis ◽

Sabir Hussain ◽

Jérémie Béguet ◽

...

Keyword(s):

Bacterial Community ◽

Large Subunit ◽

16S Rrna Gene Sequencing ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Sequencing Analysis ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Dna And Rna

ABSTRACT Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon. IMPORTANCE Our study provides novel insights into the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes, and verifies that on-farm biobed systems are hot spots for the evolution of pesticide catabolic traits.

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