16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry.

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.

Functional analysis of the Escherichia coli zinc transporter ZitB.

The membrane transporter ZitB responsible for Zn(II) efflux in Escherichia coli was studied by site-directed mutagenesis to elucidate the function of individual amino acid residues. Substitutions of several charged or polar residues, H53, H159, D163 and D186, located in predicted transmembrane domains resulted in loss of ZitB function. In contrast, neither the amino-terminal nor the carboxy-terminal regions, both histidine-rich, were required for function.

Development of a custom 16S rRNA gene library for the identification and molecular subtyping of Salmonella enterica.

The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500 bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500 bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility.

New developments in the understanding of the cation diffusion facilitator family.

Cation diffusion facilitator (CDF) proteins are a phylogenetically ubiquitous family of intermembrane transporters generally believed to play a role in the homeostasis of a wide range divalent metal cations. CDFs are found in a host of membranes, including the bacterial cell membrane, the vacuolar membrane of both plants and yeast, and the golgi apparatus of animals. As such, they are potentially useful in the engineering of hyperaccumulative phytoremediation systems. While not yet sufficient for reliable biotechnological manipulation, characterization of this family is proceeding briskly. Experimental data suggests that CDFs are generally homodimers that use proton antiport to drive substrate translocation across a membrane. This translocation of both substrate and protons is likely mediated by a combination of histidines, aspartates, and glutamates. Functional data has suggested that CDFs are not limited to metal homeostasis roles, as some appear to be determinants in the operation of high-volume metal resistance systems, and others may facilitate cation-donation as a means of signal transduction. This review seeks to give an overview of the data prompting these conclusions, while presenting additional data whose interpretation is still contentious.

FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress.

The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.

Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli.

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.