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      Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5

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          Abstract

          Background

          Cronobacter spp. are opportunistic pathogens that can cause septicemia and infections of the central nervous system primarily in premature, low-birth weight and/or immune-compromised neonates. Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteremia. It was the aim of the current study to identify genes involved in serum tolerance in a selected Cronobacter sakazakii strain of clinical origin.

          Results

          Screening of 2749 random transposon knock out mutants of a C. sakazakii ES 5 library for modified serum tolerance (compared to wild type) revealed 10 mutants showing significantly increased/reduced resistance to serum killing. Identification of the affected sites in mutants displaying reduced serum resistance revealed genes encoding for surface and membrane proteins as well as regulatory elements or chaperones. By this approach, the involvement of the yet undescribed Wzy_C superfamily domain containing coding region in serum tolerance was observed and experimentally confirmed. Additionally, knock out mutants with enhanced serum tolerance were observed. Examination of respective transposon insertion loci revealed regulatory (repressor) elements, coding regions for chaperones and efflux systems as well as the coding region for the protein YbaJ. Real time expression analysis experiments revealed, that knock out of the gene for this protein negatively affects the expression of the fimA gene, which is a key structural component of the formation of fimbriae. Fimbriae are structures of high immunogenic potential and it is likely that absence/truncation of the ybaJ gene resulted in a non-fimbriated phenotype accounting for the enhanced survival of this mutant in human serum.

          Conclusion

          By using a transposon knock out approach we were able to identify genes involved in both increased and reduced serum tolerance in Cronobacter sakazakii ES5. This study reveals first insights in the complex nature of serum tolerance of Cronobacter spp.

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          Most cited references26

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          Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov.

          [Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA-DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA-DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544(T) (=NCTC 11467(T))]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77(T) (=LMG 23826(T)=DSM 18702(T))]; Cronobacter turicensis sp. nov. [type strain z3032(T) (=LMG 23827(T)=DSM 18703(T))]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329(T) (=CIP 103581(T))]; Cronobacter dublinensis sp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515(T) (=LMG 23824=DSM 18706(T))], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464(T) (=LMG 23825(T)=DSM 18707(T))].
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            Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients.

            A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330(T), isolated from spiced meat purchased in Slovakia), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529(T) and 731, isolated from water and a leg infection, respectively) and two previously assigned to Cronobacter turicensis (strains 96 and 1435, isolated from onion powder and rye flour, respectively) was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). 16S rRNA gene sequence analysis and MLSA showed that strain 1330(T) formed an independent phylogenetic lineage in the MLSA, with Cronobacter dublinensis LMG 23823(T) as the closest neighbour. DNA-DNA reassociation and phenotypic analysis revealed that strain 1330(T) represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed (type strain 1330(T) = CECT 7863(T) = LMG 26250(T)). Strains NCTC 9529(T), 731, 96 and 1435 clustered together within an independent phylogenetic lineage, with C. turicensis LMG 23827(T) as the closest neighbour in the MLSA. DNA-DNA reassociation and phenotypic analysis confirmed that these strains represent a novel species, for which the name Cronobacter universalis sp. nov. is proposed (type strain NCTC 9529(T) = CECT 7864(T) = LMG 26249(T)).
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              RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA.

              In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.
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                Author and article information

                Contributors
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2013
                15 February 2013
                : 13
                : 38
                Affiliations
                [1 ]Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland
                Article
                1471-2180-13-38
                10.1186/1471-2180-13-38
                3621496
                23414256
                cf4085c0-5ca2-4ebc-8aaa-25d3b763105e
                Copyright © 2013 Schwizer et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 October 2012
                : 14 February 2013
                Categories
                Research Article

                Microbiology & Virology
                cronobacter sakazakii es5,clinical isolate,serum tolerance,tn5-mutagenesis,identification,pcr,complementation,expression analysis

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