Genetic and Phenotypic Diversity of Morganella morganii Isolated From Cheese

The bacterium Morganella morganii can produce the biogenic amines (BA) cadaverine, putrescine, and histamine in vitro and is responsible for high histamine concentrations in fish products. These BA can have toxic effects upon ingestion and are undesired in food. The purpose of this study was to characterize the phenotype and genotype of 11 M. morganii isolated from cheese in regard to the BA formation. In addition, we investigated the phylogeny, trehalose fermentation ability, and antibiotic resistance of the cheese isolates. To do so, we sequenced their genomes using both long and short read technologies. Due to the presence of the trehalose operon and the ability to ferment trehalose, the cheese isolates can be assigned to the subsp. sibonii. Comparative genomics with public available M. morganii genomes shows that the genomes of the cheese isolates cluster together with other subsp. sibonii genomes. All genomes between subsp. morganii and subsp. sibonii are separated by an average nucleotide identity (ANI) of less than 95.0%. Therefore, the subspecies could represent two distinct species. Nine of the strains decarboxylated lysine yielding cadaverine in vitro. This metabolic activity is linked to a previously unknown gene cluster comprising genes encoding a lysine-tRNA ligase (lysS), an HTH-transcriptional regulator (argP), a cadaverine-lysine antiporter (cadB), and a lysine decarboxylase (cadA). The formation of putrescine is linked to the speF gene encoding an ornithine decarboxylase. The gene is disrupted in five strains by an insertion sequence, and these strains only exhibit a weak putrescine production. Antimicrobial susceptibility profiling revealed that all cheese strains are resistant to tetracycline, chloramphenicol, tigecycline, colistin, and ampicillin. These phenotypes, except for colistin which is intrinsic, could be linked to antimicrobial resistance genes located on the chromosome.

Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

Diversity and Mobility of Integrative and Conjugative Elements in Bovine Isolates of Streptococcusagalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

ABSTRACT Bovine isolates of S treptococcus agalactiae (n = 76), S treptococcus dysgalactiae subsp. dysgalactiae (n = 32), and S treptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNALys) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

Proviral Structure, Chromosomal Location, and Expression of HERV-K-T47D, a Novel Human Endogenous Retrovirus Derived from T47D Particles

ABSTRACT We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408–6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3′ long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag,prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.