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Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

South Carolina coastal and inland wetlands are continentally important to resident, migrating, and wintering dabbling ducks (Anatini), diving and sea ducks (Aythini, Mergini, Oxyurini), pelagic waterbirds (Anhingidae, Laridae, Pelicanidae, and Phalcrocoracidae), and wading birds (Ardeidaie, Ciconiidae, Threskiornithidae). Our goal was to model wetland selection of these waterbird guilds in South Carolina during autumn–winter to determine habitat preferences and guide wetland conservation and restoration amid sea‐level rise and other coastal pressures. We conducted aerial surveys and recorded waterbird occurrence and relative abundance in winters 2017–2019. We modeled waterbird–habitat relationships relative to managed and unmanaged coastal and inland wetlands, legally protected conservation lands (e.g. federal, state, and private easements), and habitat diversity. Waterbirds selected a variety of wetlands emphasizing the importance of wetland diversity within habitat complexes. However, only managed tidal impoundments (MTIs) and protected conservation lands were selected across all waterbird guilds. Results suggest these are contemporary keystone habitats for promoting wintering waterbird abundance and diversity in South Carolina and other South Atlantic coastal regions. To compensate for future coastal wetland loss while facilitating the current and future needs of migrating and wintering waterbirds, we recommend: (1) identification and continued management of MTIs resilient to sea‐level rise; (2) strategic planning and partnerships for land acquisition and legal protections inland; and (3) land protection networks between extant coastal and inland sites designated for future construction and emigration of decommissioned MTIs. Stakeholder and partner engagement is paramount to prioritize resource allocation for the restoration, construction, and decommissioning of coastal and inland wetlands. [ABSTRACT FROM AUTHOR]

Early-phase transmission (EPT) is a recently described model of plague transmission that explains the rapid spread of disease from flea to mammal host during an epizootic. Unlike the traditional blockage-dependent model of plague transmission, EPT can occur when a flea takes its first blood meal after initially becoming infected by feeding on a bacteraemic host. Blockage of the flea gut results from biofilm formation in the proventriculus, mediated by the gene products found in the haemin storage (hms) locus of the Yersinia pestis chromosome. Although biofilms are required for blockage-dependent transmission, the role of biofilms in EPT has yet to be determined. An artificial feeding system was used to feed Xenopsylla cheopis and Oropsylla montana rat blood spiked with the parental Y. pestis strain KIM5(pCD1)+, two different biofilm-deficient mutants ([delta]hmsT, [delta]hmsR), or a biofilm-overproducer mutant ([delta]hmsP). Infected fleas were then allowed to feed on naive Swiss Webster mice for 1-4 days after infection, and the mice were monitored for signs of infection. We also determined the bacterial loads of each flea that fed upon naive mice. Biofilm-defective mutants transmitted from X. cheopis and O. montana as efficiently as the parent strain, whereas the EPT efficiency of fleas fed the biofilm-overproducing strain was significantly less than that of fleas fed either the parent or a biofilm-deficient strain. Fleas infected with a biofilm-deficient strain harboured lower bacterial loads 4 days post-infection than fleas infected with the parent strain. Thus, defects in biofilm formation did not prevent flea-borne transmission of Y. pestis in our EPT model, although biofilm overproduction inhibited efficient EPT. Our results also indicate, however, that biofilms may play a role in infection persistence in the flea. DOI 10.1099/mic.0.037952-0

Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a [delta]pgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal. DOI 10.1099/mic.0.037945-0

The Yersinia pestis [Hms.sup.+] phenotype is a manifestation of biofilm formation that causes adsorption of Congo red and haemin at 26[degrees]C but not at 37[degrees]C. This phenotype is required for blockage of the proventricular valve of the oriental rat flea and plays a role in transmission of bubonic plague from fleas to mammals. Genes responsible for this phenotype are located in three separate operons, hmsHFRS, hmsT and hmsP. HmsH and HmsF are outer membrane (OM) proteins, while the other four Hms proteins are located in the inner membrane. According to the Hidden Markov Method-based predictor, HmsH has a large N terminus in the periplasm, a [beta]-barrel structure with 16 [beta]-strands that traverse the OM, eight surface-exposed loops, and seven short turns connecting the [beta]-strands on the periplasmic side. Here, we demonstrate that HmsH is a heat-modifiable protein, a characteristic of other [beta]-barrel proteins, thereby supporting the bioinformatics analysis. Alanine scanning mutagenesis was used to identify conserved amino acids in the HmsH-like family that are critical for the function of HmsH in biofilm formation. Of 23 conserved amino acids mutated, four residues affected HmsH function and three likely caused protein instability. We used formaldehyde cross-linking to demonstrate that HmsH interacts with HmsF but not with HmsR, HmsS, HmsT or HmsP. Loss-of-function HmsH variants with single alanine substitutions retained their [beta]-structure and interaction with HmsF. Finally, using a polar hmsH: : mini-kan mutant, we demonstrated that biofilm development is not important for the pathogenesis of bubonic or pneumonic plague in mice. DOI 10.1099/mic.0.036640-0

Yersinia pestis cells were grown in vitro at 26 and 37[degrees]C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37[degrees]C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small [beta]-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37[degrees]C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26[degrees]C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26[degrees]C, but not at 37[degrees]C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658-y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a [M.sub.r] range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.

Yersinia pestis genomes contain genes homologous to the aerobactin receptor (iutA) and biosynthetic genes (iucABCD) as well as the ferric hydroxamate uptake system (fhuCDB) of Escherichia coli However, iucA is disrupted by a frameshift mutation. An E. coli strain carrying the cloned Y. pestis aerobactin region was unable to produce aerobactin, but could use the siderophore as an iron source. Repair of the frameshift mutation in iucA did not allow aerobactin production in E. coli or Y. pestis. In contrast, a Y. pestis strain with a plasmid encoding the iucABCD-iutA genes from Shigella flexneri or pColV-K30 did produce and secrete the siderophore. In addition, Yersinia pseudotuberculosis PB1, which encodes the iucABCD-iutA locus without the K pestis-specific frameshift mutation, also failed to produce aerobactin. The Y. pestis fhuCDB operon, encoding an ABC transporter for a range of hydroxamate siderophores, was able to complement a strain of E. coli with a transposon insertion in fhuC, allowing utilization of aerobactin and ferrichrome. Y. pestis KIM6, a strain deficient in the production of the siderophore yersiniabactin, was able to use both the ferrichrome and the aerobactin siderophores as a source of iron. Mutations in iutA or the fhu operon abolished the ability of KIM6 to use aerobactin. Mutations in the fhu operon, but not in iutA, affected the ability of KIM6 to use ferrichrome. This demonstrates that Y. pestis uses both ferrichrome and aerobactin, but has lost the ability to synthesize aerobactin.

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a [beta]- barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

We provide the first evidence for a link between polyamines and biofilm levels in Yersinia pestis, the causative agent of plague. Polyamine-deficient mutants of Y. pestis were generated with a single deletion in speA or speC and a double deletion mutant. The genes speA and speC code for the biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase, respectively. The level of the polyamine putrescine compared to the parental [speA.sup.+] [speC.sup.+] strain (KIM6+) was depleted progressively, with the highest levels found in the Y. pestis AspeC mutant (55% reduction), followed by the AspeA mutant (95% reduction) and the AspeA AspeC mutant (>99% reduction). Spermidine, on the other hand, remained constant in the single mutants but was undetected in the double mutant. The growth rates of mutants with single deletions were not altered, while the AspeA AspeC mutant grew at 65% of the exponential growth rate of the [speA.sup.+] [speC.sup.+] strain. Biofilm levels were assayed by three independent measures: Congo red binding, crystal violet staining, and confocal laser scanning microscopy. The level of biofilm correlated to the level of putrescine as measured by high-performance liquid chromatography-mass spectrometry and as observed in a chemical complementation curve. Complementation of the AspeA AspeC mutant with speA showed nearly full recovery of biofilm to levels observed in the [speA.sup.+] [speC.sup.+] strain. Chemical complementation of the double mutant and recovery of the biofilm defect were only observed with the polyamine putrescine.

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.

Yersinia pestis is a facultative intracellular bacterial pathogen that can replicate in macrophages. Little is known about the mechanism by which Y. pestis replicates in macrophages, and macrophage defense mechanisms important for limiting intracellular survival of Y. pestis have not been characterized. In this work, we investigated the ability of Y. pestis to replicate in primary murine macrophages that were activated with IFN-[gamma] Y. pestis was able to replicate in macrophages that were activated with IFN-[gamma] after infection (postactivated). A region of chromosomal DNA known as the pigmentation (pgm) locus was required for replication in postactivated macrophages, and this replication was associated with reduced nitric oxide (NO) levels but not with reduced inducible NO synthase (iNOS) expression. Y. pestis [DELTA]pgm replicated in iNO[S.sup.-/-] macrophages that were postactivated with IFN-[gamma], suggesting that killing of [DELTA]pgm Y. pestis is NO-dependent. A specific genetic locus within pgm, which shares similarity to a pathogenicity island in Salmonella, was shown to be required for replication of Y. pestis and restriction of NO levels in postactivated macrophages. These data demonstrate that intracellular Y. pestis can evade killing by macrophages that are exposed to IFN-[gamma] and identify a potential virulence gene encoded in the pgm locus that is required for this activity. intracellular replication | nitric oxide

The complete genome sequence of Yersinia pestis KIM is presented. The 4.6-Mb genome encodes 4,198 open reading frames and bear broad similarity to Escherichia coli K-12 sequence with respect to the origin, terminus, and DNA replication protein encoding genes. The KIM-specific island genes encode pathogenicity proteins.

A study was conducted to chracterize the functions of Yersinia pestis YbtE and high molecular weight protein 2 (HMWP2) enzymes in the biosynthesis of yersiniabactin. Polymerase chain reaction was carried out to amplify the Y. pestis ybtE gene while cultures of Escherichia coli strain were prepared at 37 degrees C. Results indicated that the heterocyclization capacity of HMWP2 1-1491 correlated with salicyl-transferring and thiazoline-forming activities.

The transduction of the Escherichia coli SAB11 deficient in the biosynthesis of the siderophore enterobactin was implemented utilizing a cosmid library obtained from the genomic DNA of Yersinia pestis KIM 6+. Recombinant plasmids containing a common 13-kb BamHI fragment were then derived from SAB11 transductants. The results of transposon mutagenesis and subcloning found that a 5.6-kb region was important for stimulating the growth of SAB11.

Sequencing of a region from the pgm locus of Yersinia pestis KIM6+ which transmits sensitivity to the bacteriocin pesticin to certain strains of Y. pestis and Escherichia coli showed that the Y. Pestis sequence is almostidentical to the pesticin receptor from Yersinia enterocolitica and is homologous to other outer membrane TonB-dependent proteins. The protein manufactured by the irp2 mutant suggests that HMWP2 may be indirectly involved for the maximal expression of the pesticin receptor.

Yersinia pestis mutation, prepared by insertional disruption of the fur open reading frame, affects the expression of many iron-regulated proteins. These Y. pestis cells become more sensitive to bacteriocin pestin with mutations in fur. There is an increase in late log phase spontaneous nonpigmented chromosomal deletion mutants and a block in the growth of the Y. pestis fur mutant in excess of iron.

Abstract Background The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C). Results Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells. Conclusions Many protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26°C and 37°C), there was little evidence for temperature-specific adaptation processes to iron starvation.

Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.