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Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

ABSTRACT: Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal ?-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1?A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1?A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

SUBMITTER: Singhal N

PROVIDER: S-EPMC7311522 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

Singhal Neelja N Pandey Deeksha D Singh Nambram Somendro NS Kumar Manish M Virdi Jugsharan Singh JS

Scientific reports 20200623 1

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal β-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in ...[more]

PMID: 32576927

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Project description:BackgroundSuperoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism.Methodology/principal findingsThis study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB.Conclusions/significanceThis is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.

Project description:We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5'-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase -specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

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Dataset Information

Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

Publications

Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

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