Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

<p>Abstract</p> <p>Background</p> <p>Urease is a virulence factor that plays a role in the resistance of <it>Brucella </it>to low pH conditions, both <it>in vivo </it>and <it>in vitro</it>. <it>Brucella </it>contains two separate urease gene clusters, <it>ure1 </it>and <it>ure2</it>. Although only <it>ure1 </it>codes for an active urease, <it>ure2 </it>is also transcribed, but its contribution to <it>Brucella </it>biology is unknown.</p> <p>Results</p> <p>Re-examination of the <it>ure2 </it>locus showed that the operon includes five genes downstream of <it>ureABCEFGDT </it>that are orthologs to a <it>nikKMLQO </it>cluster encoding an ECF-type transport system for nickel. <it>ureT </it>and <it>nikO </it>mutants were constructed and analyzed for urease activity and acid resistance. A non-polar <it>ureT </it>mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The <it>nikO </it>mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium.</p> <p>Conclusions</p> <p>Based on these results, we concluded that the operon <it>ure2 </it>codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the <it>ure1 </it>operon.</p

Evaluation of algaecide effectiveness of five different oxidants applied on harmful phytoplankton

Harmful algal blooms (HABs) in coastal areas similarly impact both ecosystems and human health. The translocation of phytoplankton species via maritime transport can potentially promote the growth of HABs in coastal systems. Accordingly, ballast water must be disinfected. The main goal of this study is to assess the effectiveness of different emerging biocides, including H2O2, peracetic acid (PAA), peroxymonosulfate (PMS), and peroxydisulfate (PDS). The effectiveness of these biocides is compared with that of conventional chlorination methods. Their effects on two ichthyotoxic microalgae with worldwide distribution, i.e., Prymnesium parvum and Heterosigma akashiwo, are examined. To ensure the prolonged effectiveness of the different reagents, their concentration–response curves for 14 days are constructed and examined. The results suggest a strong but shorter effect by PMS (EC50 = 0.40–1.99 mg·L-1) and PAA (EC50 = 0.32–2.70 mg·L-1), a maintained effect by H2O2 (EC50 = 6.67–7.08 mg·L-1), and a negligible effect by PDS. H. akashiwo indicates higher resistance than P. parvum, except when H2O2 is used. Based on the growth inhibition performance and consumption of the reagents as well as a review of important aspects regarding their application, using H2O2, PAA, or PMS can be a feasible alternative to chlorine-based reagents for inhibiting the growth of harmful phytoplankton.This work has been co-funded by the 2014–2020 ERDF Operational Programme and by the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia (Spain). Project Ref.: FEDER-UCA18–108023. This work is part of the project TED2021–130994B-C31; TED2021–130994B-C33 and Grant IJC2020–042741-I funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR

Epitopes for Multivalent Vaccines Against Listeria, Mycobacterium and Streptococcus spp: A Novel Role for Glyceraldehyde-3-Phosphate Dehydrogenase

The glycolytic enzyme and bacterial virulence factor of Listeria monocytogenes, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Lmo2459), ADP-ribosylated the small GTPase, Rab5a, and blocked phagosome maturation. This inhibitory activity localized within the NAD binding domain of GAPDH at the N-terminal 1?22 peptides, also conferred listeriosis protection when used in dendritic cell-based vaccines. In this study, we explore GAPDH of Listeria, Mycobacterium, and Streptococcus spp. taxonomic groups to search for epitopes that confer broad protection against pathogenic strains of these bacteria. GAPDH multivalent epitopes are selected if they induce inhibitory actions and wide-ranging immune responses. Proteomic isolation of GAPDH from dendritic cells infected with Listeria, Mycobacterium, or Streptococcus confirmed similar enzymatic, Rab5a inhibitory and immune stimulation abilities. We identified by bioinformatics and functional analyses GAPDH N-terminal 1?22 peptides from Listeria, Mycobacterium, and Streptococcus that shared 95% sequence homology, enzymatic activity, and B and T cell immune domains. Sera obtained from patients or mice infected with hypervirulent pathogenic Listeria, Mycobacterium, or Streptococcus presented high levels of anti-GAPDH 1?22 antibodies and Th2 cytokines. Monocyte derived dendritic cells from healthy donors loaded with GAPDH 1?22 peptides from Listeria, Mycobacterium, or Streptococcus showed activation patterns that correspond to cross-immunity abilities. In summary, GAPDH 1?22 peptides appeared as putative candidates to include in multivalent dendritic based vaccine platforms for Listeria, Mycobacterium, or Streptococcus

Evaluation of the effects of erythritol on gene expression in Brucella abortus

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORF's constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol

Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921

Letter to the Editor.-- El pdf es la versión post-print.This work was supported by grant PI05/0894 from ‘Fondo de investigación Sanitaria’ of the Spanish Ministry of Science and Innovation.Peer Reviewe

Identification of a Streptogramin A Acetyltransferase Gene in the Chromosome of Yersinia enterocolitica

Streptogramins are polypeptide antibiotics inhibiting protein synthesis by the prokaryotic ribosome. Gram-positive organisms are susceptible to streptogramins, while most gram-negative bacteria are intrinsically resistant. We have found a genomic fragment from a Yersinia enterocolitica isolate with an open reading frame coding for a polypeptide similar to the virginiamycin acetyltransferases found in various plasmids from gram-positive bacteria. The susceptible Escherichia coli strain DB10 was transformed to resistance to the type A streptogramins and to mixed (A + B) streptogramins upon introduction of a plasmid containing that gene. In addition, we showed streptogramin acetylating activity in vitro dependent on the presence of the Y. enterocolitica sat gene. Southern blot hybridization experiments showed that the sat gene was present in all the Y. enterocolitica isolates examined. These data together show that the gene in the Y. enterocolitica chromosome encoded an active streptogramin acetyltransferase. The deduced sequence of the Y. enterocolitica Sat protein was close to those of sat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin

Tandem Amplification of a 28-Kilobase Region from the Yersinia enterocolitica Chromosome Containing the blaA Gene

Most Yersinia enterocolitica strains are resistant to β-lactam antibiotics due to the production of one or two chromosomally encoded β-lactamases. Strain Y56 is a Y. enterocolitica O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A β-lactamase. To select mutants with increased levels of resistance to β-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 μg of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a blaA-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the blaA gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the blaA locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event

The exploration of Brucella transcriptome: From the ORFeome to RNAseq

El pdf es la versión post-print.In this chapter we will analyze the results available on the characterization of the Brucella transcriptome. After a summary of earlier work on transcription, two technical approaches will be mainly described, on one side the use microarrays, specially that derived from the Brucella ORFeome that allows hybridization with mRNA derived cDNA to determine the relative abundance of transcripts from each B. melitensis ORF. On the other, RNAseq, consisting in the massive sequencing of cDNA libraries derived from mRNA obtained from B. abortus grown in culture medium. Sequencing with the Illumina Genome-Analyzer II platform, produced 3 millions of 35 nt long reads that annealed with single copy coding regions of the genome. This allowed a good coverage for every CDS and produced a new dataset on the transcription of Brucella. We obtained a good correlation for the set of highly expressed genes from the microarrays and confirmed the observations obtained on the asymmetry between chromosome transcription. Preliminary conclusions on intracellular transcription have been drawn from RT-PCR on selected candidate genes and from microarray datasets obtained from virulence related conditions.The RNAseq derived data allowed more versatile data mining giving some new details on transcription from pseudogenes or intergenic regions.Peer Reviewe