The Majority of Typhoid Toxin-Positive Salmonella Serovars Encode ArtB, an Alternate Binding Subunit.

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, an A2B5 toxin known as the typhoid toxin (TT), was recently identified among a variety of S. enterica serovars. While past studies have shown that some serovars encode both the TT (active subunits CdtB and PltA and binding subunit PltB) and a second binding subunit (ArtB), these serovars were thought to be the exception. Here, we show that genes encoding the TT are detected in more than 100 serovars representing distinct phylogenetic lineages of S. enterica subsp. enterica, although clade B and section Typhi are significantly more likely to encode TT genes than serovars from other clades. Furthermore, we show that 81% of these TT-positive serovars also encode artB, suggesting that the cooccurrence of both toxin binding subunits is considerably more common than previously thought. A combination of in silico modeling, bacterial two-hybrid system screening, and tandem affinity purification (TAP) of toxin subunits suggests that ArtB and PltB interact in vitro, at least under some growth conditions. While different growth conditions yielded slightly higher transcript abundances of artB and pltB, both genes had their highest relative transcript abundances when Salmonella was grown under low-Mg2+ conditions, suggesting that ArtB and PltB may compete for inclusion in the TT. Together, our results suggest that ArtB likely plays an important and previously underappreciated role in the biology of the TT produced by typhoidal and nontyphoidal SalmonellaIMPORTANCE While previous reports had suggested that the typhoid toxin (TT) could potentially use ArtB as an alternate binding subunit, this was thought to play a minor role in the evolution and biology of the toxin. In this study, we establish that both TT genes and artB are widespread among Salmonella enterica subsp. enterica, suggesting that TT likely plays a broader role in Salmonella virulence that extends beyond its proposed role in typhoid fever. Furthermore, our data suggest the selective maintenance of both toxin binding subunits, which may compete for inclusion in the holotoxin. Last, our data support the importance of characterizing diverse nontyphoidal Salmonella (NTS) serovars, as the presence of classically defined typhoidal virulence factors among NTS serovars continues to challenge the typhoid-nontyphoid Salmonella paradigm.

Transcriptional profiling of the L. monocytogenes PrfA regulon identifies six novel putative PrfA-regulated genes.

The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows: (i) prfA* and WT; (ii) WT and ∆prfA and (iii) prfA* and ∆prfA. Significantly higher transcript levels in the induced WT or constitutively active PrfA* were identified for 18 genes and 2 ncRNAs in at least one of the three comparisons. These genes included: (i) 10/12 of the genes previously identified as directly PrfA-regulated; (ii) 2 genes previously identified as PrfA-regulated, albeit likely indirectly; and (iii) 6 genes newly identified as PrfA-regulated, including one (LMRG_0 2046) with a σA-dependent promoter and PrfA box located within an upstream open reading frame. LMRG_0 2046, which encodes a putative cyanate permease, is reported to be downregulated by a σB-dependent anti-sense RNA. This newly identified overlap between the σB and PrfA regulons highlights the complexity of regulatory networks important for fine-tuning bacterial gene expression in response to the rapidly changing environmental conditions associated with infection.

Assessment of TREC, KREC and telomere length in long-term survivors after allogeneic HSCT: the role of GvHD and graft source and evidence for telomere homeostasis in young recipients.

Reconstitution of the adaptive immune system following allogeneic hematopoietic stem cell transplantation is crucial for beneficial outcome and is affected by several factors, such as GvHD and graft source. The impact of these factors on immune reconstitution has been thoroughly investigated during the early phase after transplantation. However, little is known about their long-term effect. Similarly, leukocyte telomere length (TL) shortening has been reported shortly after transplantation. Nevertheless, whether TL shortening continues in long-term aspect is still unsettled. Here, we assessed T-cell receptor excision circle (TREC), kappa deleting recombination excision circle (KREC) and leukocyte TL in recipients and donors several years post transplantation (median 17 years). Our analysis showed that, recipients who received bone marrow (BM) as the graft source have higher levels of both TREC and KREC. Also, chronic GvHD affected TREC levels and TL but not KREC levels. Finally, we show that recipient's TL was longer than respective donors in a group of young age recipients with high KREC levels. Our results suggest that BM can be beneficial for long-term adaptive immune recovery. We also present supporting evidence for recipient telomere homeostasis, especially in young age recipients, rather than telomere shortening.

Identification of target promoters for the Bacillus subtilis extracytoplasmic function sigma factor, sigma W.

The Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) sigma factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, PW. Using a consensus-based search procedure, we have identified 15 operons preceded by promoters similar in sequence to PW. At least 14 of these promoters are dependent on sigma W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays and nucleotide resolution start site mapping. We conclude that sigma W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The sigma W regulon includes a penicillin binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides. We suggest that sigma W activates a large stationary-phase regulon that functions in detoxification, production of anti-microbial compounds or both.

Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake.

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC-deficient mutant is also compromised in the production and utilization of pyoverdine, the high-affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane-spanning regions with three periplasmic loops. Site-directed mutagenesis was used in order to assess the importance of some periplasm-exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His-61, Val-62 and Pro-63 in the first periplasmic loop, and Leu-184, His-185 and Gln-186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp-115 and Gly-116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp-127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp-126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis.

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA. A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region. A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation. Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.

A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens.

Detection and differentiation of microbial siderophores by isoelectric focusing and chrome azurol S overlay.

Siderophores are microbial, low molecular weight iron-chelating compounds. Fluorescent Pseudomonads produce different, strain-specific fluorescent siderophores (pyoverdines) as well as non-fluorescent siderophores in response to low iron conditions. We present an isoelectric focusing method applicable to unpurified as well as to purified pyoverdine samples where the fluorescent siderophores are visualized under UV illumination. Siderophores from different Pseudomonas sp., amongst which are P. aeruginosa, P. fluorescens and P. putida, including egg yolk, rhizospheric and clinical isolates as well as some derived Tn5 mutants were separated by this technique. Different patterns could be observed for strains known to produce different siderophores. The application of the chrome azurol S assay as a gel overlay further allows immediate detection of non-fluorescent siderophores or possibly degradation products with residual siderophore activity. The method was also applied to other microbial siderophores such as deferrioxamine B.

A field survey on child health in a rural community in Egypt.

A field survey in a village was carried out to study child health in a rural area. The vaccination scheme was found to be inadequate and the value of health education is stressed. Parasitic infestation was found to be higher than urban areas but significantly lower than 1959 and the latter is attributed to a safer water supply. Growth and development lagged behing the whole national standards particularly during the 1 - 6 years age period. Health care for the pre-school age, taking advantage of co-operation from medical faculties and local health authorities, is recommended.