OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem−1 and weaker phem−2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the −35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem−1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5′-untranslated region (5′-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts

Transcriptional Organization of the Stability Module of BroadHost-Range Plasmid RA3, from the IncU Group

The broad-host-range (BHR) conjugative plasmids have developed diverse adaptive mechanisms defining the range of their promiscuity. The BHR conjugative RA3 plasmid, the archetype of the IncU group, can transfer between, replicate in, and be maintained in representatives of Alpha-, Beta-, and Gammaproteobacteria. Its stability module encompasses ten open reading frames (ORFs) apparently organized into five operons, all transcribed in the same direction from several strong promoters that are tightly regulated either by autorepressors or by global plasmidencoded regulators. In this paper, we demonstrate that owing to an efficient RNA polymerase (RNAP) read-through, the transcription from the first promoter, orf02p, may continue through the whole module. Moreover, an analysis of mRNA produced from the wild-type (WT) stability module and its deletion variants deprived of particular internal transcription initiation sites reveals that in fact each operon may be transcribed from any upstream promoter, giving rise to multicistronic transcripts of variable length and creating an additional level of gene expression control by transcript dosage adjustment. The gene expression patterns differ among various hosts, indicating that promoter recognition, regulation, and the RNAP read-through mechanisms are modulated in a species-specific manner

The effect of age and ultimate pH value on selected quality traits of meat from wild boar.

The meat from hunted wild boar juveniles (N=18) and yearlings (N=17), was analysed to assess the influence of age, and to assess the ultimate pH value on selected quality traits. The analysed meat of 55.56 % of the juveniles and 64.71 % of the yearlings was characterised with normal pH. The pH had been measured 24 h and 48 h post-mortem. More cases of high ultimate pH (pHu>5.8) and high maximal pH (about 6.2) have been noted in the meat of younger animals compared to older ones. We found no effect of pHu on the colour coordinates of analysed wild boar meat. A slight effect of age was observed for the L* coordinate. The post-mortem time was the most important factor influencing meat colour (L*, b* and hue-angle). A high pHu was related to lower drip loss (P=0.001), lower percentage of free water (P=0.036), lower cooking loss (P=0.001), and lower plasticity (P=0.042). The meat from juveniles showed higher plasticity than meat from yearlings. Summing up, both the pHu level and the age of wild boars may affect some qualitative patterns of meat, changing the technological usability of this raw animal product.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning.

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn<i>3</i>-Derived Inverted-Repeat Miniature Elements (TIMEs)

<div><p>Functional transposable elements (TEs) of several <i>Pseudomonas</i> spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS<i>3</i>, IS<i>5</i>, IS<i>L3</i>, IS<i>30</i> and IS<i>1380</i>), (ii) isoforms of two Tn<i>3</i>-family transposons – Tn<i>5563</i>a and Tn<i>4662</i>a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn<i>5563</i>a and many other Tn<i>3</i>-family transposons. The simplest elements of this type, designated TIMEs (Tn<i>3</i> family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of <i>Pseudomonas</i> spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a <i>res</i> site typical for many Tn<i>3</i>-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.</p></div

Possible mechanism for the generation of diverse non-autonomous and autonomous elements of the Tn<i>3</i> transposon family.

<p>The elements of the Tn3 family originate from progenitor insertion sequences (IS). Tns – diverse autonomous non-composite or composite transposons generated by acquisition of foreign DNA and mobilization for transposition of genomic DNA results. TIME - non-autonomous elements resulting from a reduction in the number of transposon-encoded genes, able to form mosaic elements resembling non-composite and composite Tns in structure. Elements representative of the different types are shown as examples: IS<i>1071</i> (accession no. M65135), Tn<i>3434</i> (accession no. AY232820), Tn<i>5393</i> (accession no. M96392), Tn<i>Ppa1</i> (accession no. DQ149577) and Tn<i>5271</i> (accession no. U18133).</p

Genetic structure of <i>Pseudomonas</i> spp. plasmids containing non-autonomous TEs of the Tn<i>3</i> transposon family.

<p>Circular plasmids pZM1P1 (A) and pLM8P2 (B) originate from <i>Pseudomonas</i> spp. strains ZM1 and LM8, respectively. Predicted genetic modules involved in plasmid replication (REP), mobilization for conjugal transfer (MOB) and toluene resistance (TtgGHI efflux pump - truncated operon) are boxed and appropriately labeled. Predicted coding regions are represented by thick arrows indicating the direction of transcription. Broken arrows indicated truncated genes (<i>ttgH</i> and <i>orf5</i> of pLM8P2). Shaded areas connect DNA regions of pZM1P1 included in non-autonomous TIME-COMP transposons. Note that TIMEs are not drawn to scale. Thin black arrows above TIMEs indicate the orientation of the elements. A plot of the G+C content of pZM1P1 is shown above the structure diagram and the average G+C value is given to the right.</p

Predicted secondary structures of TIMEs at the RNA level.

<p>RNA secondary structures predicted by <i>in silico</i> folding using Mfold software for TIME1 (class 1), two TIMEs representing class 3 and 4 elements shown in Fig. 3 (accession nos. AF020724 and AE016855, respectively), and the TIME-like elements ARM<i>phe</i> and IS<i>101</i> (see Discussion for details). The minimum folding energy (ΔG) of the predicted secondary structures was calculated by Mfold.</p