Ahmed Gaballa

Listeria swaminathanii UTK S2-0008, isolated from soil collected in the Nantahala National Forest in North Carolina, USA, is the only L. swaminathanii strain eligible to serve as the type strain, which is needed to achieve valid status. The previously effectively published type, L. swaminathanii FSL L7-0020 ^T , and previously described strains UTK C1-0015 and UTK C1-0024 do not conform to the International Code of Nomenclature of Prokaryotes’ rules for type strains. Additionally, the currently designated type strain (FSL L7-0020 ^T = ATCC TSD-239 ^T ) is an atypical representative of L. swaminathanii as it is the only strain lacking catalase activity. Therefore, it is proposed to reassign the type to L. swaminathanii (UTK S2-0008 ^T = CCUG 77280 ^T = LMG 33255 ^T ). Whole-genome sequence-based average nucleotide identity (ANI) showed that this strain clustered with the three previously described L. swaminathanii strains (FSL L7−0020 = ATCC TSD-239, UTK C1-0015, and UTK C1-0024; pairwise ANI ranged from 98.71% to 98.83%). All four strains, including the one described here, could not be classified as any validly published Listeria species and showed the highest similarity to Listeria marthii (maximum ANI of 93.92%, in silico DNA-DNA hybridization of 56.2%). L. swaminathanii exhibits the phenotypic characteristics that are currently expected of the Listeria sensu stricto species. This species lacks phenotypic characteristics associated with Listeria pathogenicity (non-hemolytic and negative for phosphatidylinositol-specific phospholipase C activity); the genomes lack genes associated with virulence (all genes found on the Listeria pathogenicity island 1 [LIPI-1], as well as the internalin genes inlA and inlB ), which support L. swaminathanii is nonpathogenic. IMPORTANCE: The genus Listeria includes species of significant relevance to food safety, environmental microbiology, and public health. Accurate species identification is critical because misidentification of nonpathogenic species as pathogenic ones can lead to unnecessary recalls and regulatory complications. The validation of Listeria swaminathanii sp. nov. will ensure that this species is formally recognized and has a type strain (UTK S2-0008 ^T ) that is representative of the species. This work strengthens diagnostic accuracy by enabling the inclusion of this species in reference databases and inclusivity studies, reducing the risk of false identification. Furthermore, the identification and characterization of Listeria swaminathanii sp. nov. expands our understanding of the genetic and ecological diversity within the genus Listeria , particularly among soil-dwelling strains.

Bacillus subtilis CpgA ( c ircularly p ermuted G TPase) is a ribosome assembly GTPase that has a secondary function as a metabolite proofreading enzyme. CpgA hydrolyzes 4-phosphoerythronate, a toxic metabolite produced from erythrose-4-phosphate by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a ∆ cpgA strain, carbon sources that feed into the pentose phosphate pathway trigger metabolic intoxication. This results in poor growth and increased sensitivity to antibiotics that block peptidoglycan synthesis, a process reliant on sugars from central metabolism. Here, we describe a mutation in ptsH ( ptsH -G54D) that improves growth of a ∆ cpgA strain on media containing both glucose and gluconate. The ptsH gene encodes the histidine-containing phosphocarrier protein (HPr) that functions in phosphotransferase system sugar import and gene regulation. Prior studies of HPr suggested three possible mechanisms to account for the ability of HPr-G54D to increase fitness of the ∆ cpgA strain: (i) restricting HPr-dependent uptake of glucose, (ii) reducing the GAPDH-dependent production of 4-phosphoerythronate, or (iii) decreasing expression of genes required for uptake and catabolism of gluconate. Here, we present evidence consistent with the third model: HPr-G54D improves fitness of a ∆ cpgA strain by increasing catabolite repression of the gluconate operon. Consistently, genetic suppression by HPr-G54D requires Ser46, a site of regulatory phosphorylation important for carbon catabolite repression. In addition, we demonstrate that the metabolic proofreading function of CpgA is conserved among related gram-positive bacteria. IMPORTANCE: Metabolism relies on the concerted action of hundreds of enzymes, many of which have some activity with non-canonical substrates. The resulting reactions constitute an often-ignored underground metabolism. Glyceraldehyde-3-phosphate dehydrogenase catalyzes a secondary reaction that produces 4-phosphoerythronate, a toxic dead-end metabolite. Bacillus subtilis CpgA is a widely conserved metabolite proofreading enzyme that protects cells against metabolic intoxication, which can increase antibiotic sensitivity. Loss of CpgA can be suppressed by an altered function mutation affecting the histidine-containing phosphocarrier protein (HPr). This mutant HPr protein increases carbon catabolite repression to restrict import of intoxicating gluconate. These studies highlight the ability of mutations in HPr to rewire carbon catabolism to help avoid the toxic effects of metabolic dysregulation.

Metal ions are universally essential for life and are required for critical enzymes throughout metabolism. Metalloenzymes rely on metal import and trafficking pathways for loading of the desired metal. Many microbes produce natural products that serve as metal chelators, both for their own nutrition and to serve as antimicrobials. In response to infection, our immune cells restrict bacterial growth by deploying proteins that chelate metal ions as part of nutritional immunity. Cells respond to metal depletion by the activation of pathways that prioritize metal delivery to the most essential enzymes. Dithiolopyrrolone (DTP) class natural products are prodrugs that are reduced in cells to generate a potent, dithiol-containing zinc chelator. Here, we identify the cellular reductants, bacillithiol and the FAD-dependent oxidoreductases TrxB and AhpF, that activate the DTP antibiotic thiolutin in Bacillus subtilis . Genetic studies reveal that loss of the Spx transcription factor also increases thiolutin resistance, consistent with the known role of Spx in transcriptional activation of thioredoxin reductase ( trxB ) and genes required for bacillithiol synthesis. Collectively, our results support a model in which several parallel pathways all contribute to the reductive activation of DTP class prodrugs in vivo . IMPORTANCE: Metal ion chelators (metallophores) are deployed by microbes to obtain nutrient metals, sequester excess metals, and act as antimicrobials to inhibit the growth of other organisms. Dithiolopyrrolones (DTPs) are a class of natural products that inhibit bacterial growth by the intracellular chelation of zinc and iron, two metal ions essential for growth. Thiolutin, a model DTP antibiotic, is activated by reduction inside cells and selectively chelates intracellular metals. Here, we demonstrate that the activation of the thiolutin prodrug is mediated by several parallel pathways, which greatly reduces the ability of cells to evolve antibiotic resistance. Since DTP antibiotics appear to primarily target zinc enzymes, they provide a powerful tool for exploring how cells adapt to zinc deficiency.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE: Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes . Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes . Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σ^B, σ^L, σ^H, and σ^C (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σ^B-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σ^L-dependent, and no σ^H- or σ^C-dependent genes showed FC >100. While σ^B-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σ^L appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σ^B and σ^L directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σ^B regulates multiple stress response pathways, while σ^L and σ^H positively regulate a smaller number of specific pathways.

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, a DNA-damaging toxin known as the typhoid toxin (TT), was recently characterized in >40 nontyphoidal Salmonella (NTS) serovars. Interestingly, these NTS serovars, including S. enterica subsp. enterica serovar Javiana, also encode artB , a homolog of the binding subunit (PltB) of the TT. Here, we show that ArtB and PltB compete for inclusion in the pentameric binding subunit of the TT. Using a combination of in silico modeling, a bacterial two-hybrid system expressed in S. Javiana, and tandem affinity purification (TAP) of the holotoxin subunits, we show that ArtB and PltB interact in vivo . Furthermore, binding subunits composed of homo- and heteropentamers of ArtB and PltB are able to associate with CdtB and PltA to form biologically active toxins. As artB was, (i) conserved among S. Javiana isolates, and (ii) co-expressed with pltB and cdtB under Mg ²⁺ -limiting conditions, we hypothesized that ArtB and PltB compete for inclusion in the binding subunit. Using a novel competition assay, we show that PltB outcompetes ArtB for inclusion in the binding subunit, when cultured at neutral pH. Together, our results suggest that the TT produced by S. Javiana utilizes multiple configurations of the binding subunit, representing a novel toxin form and adaptation mechanism for the AB ₅ toxin family. Our work suggests that Salmonella serovars, including S. Javiana, evolved to encode and maintain multiple binding subunits that can be used to form an active toxin, which may enhance the variety of cells, tissues, or hosts susceptible to this novel form of the TT.

Mobilized colistin resistance ( mcr ) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described ( mcr -1 to -8). Here, we describe mcr -9, a novel mcr homologue, detected in a Salmonella enterica serotype Typhimurium ( S. Typhimurium) genome using an in silico approach, followed by experimental functional analysis. The amino acid sequence of mcr -9, detected in a multidrug resistant (MDR) S. Typhimurium strain isolated from a human patient in Washington State in 2010, most closely resembled mcr -3, aligning with 64.5% amino acid identity and 99.5% coverage using translated nucleotide blast. The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2 mg/L European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. To determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host, mcr -9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG-induced promoter. Expression of mcr -9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/L colistin, albeit at a lower level when compared to mcr -3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, and Mcr-7 share a high degree of similarity at the structural level. The results of our approach indicate that mcr -9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance. Importance: Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a Highest Priority Critically Important Antimicrobial for human medicine (WHO, Critically Important Antimicrobials for Human Medicine, 5th Revision , 2017), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.