The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

Phage-associated mutator phenotype in group A streptococcus

Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5′ end of mutL correlates with a mutator phenotype (10⁻⁷ to 10⁻⁸ mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10⁻⁹ to 10⁻¹⁰ mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.     

Competition between isogenic mutS and mut+ populations of Escherichia coli K12 in continuously growing cultures

Summary Previous studies have shown that the mutT, mutH and mutL mutators of Escherichia coli have a marked advantage in competition growth with otherwise coisogenic wild-type strains. As shown in this paper the same is true for the mutS mismatch mutator. In three experiments mutS could outgrow the wild-type and had higher fitness values.     

Fructose-Asparagine Is a Primary Nutrient during Growth of Salmonella in the Inflamed Intestine

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10^−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1⁻ SPI2⁻ or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.     

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutL_E36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.     

Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression

Mutations in the chromatin remodeller‐coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention‐deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7 ^−/− model. chd7 ^−/− mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype‐based screen in chd7 ^−/− zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies.     

The 9-1-1 DNA clamp is required for immunoglobulin gene conversion

Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9^−/− and RAD17^−/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9^−/− and RAD17^−/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.     

Inefficient mismatch repair: genetic defects and down regulation

The mismatch repair system is involved in the maintenance of genomic integrity by editing DNA replication and recombination. However, although most mutations are neutral or deleterious, a mutator phenotype due to an inefficient mismatch repair may generate advantageous variants and may therefore be selected for. We review the evidence for inefficient mismatch repair due either to genetic defects in mismatch repair genes or to physiological conditions. Among natural isolates ofEscherichia coli andSalmonella enterica, about 1% are mutator bacteria, mostly deficient in mismatch repair (most of them defective in themutS gene). Characterization of mutators derived from laboratory strains led also to the isolation of mismatch repair mutants in which the most frequently found defects are inmutL andmutS. The correlation of the size of the antimutator genes with the frequency of their defective alleles amongE. coli andSalmonella strains reveals thatmutU mutants are underrepresented. Analysis of the progeny of a defined M13 phage heteroduplex DNA transfected intoE. coli cells shows that mismatch repair efficiency progressively decreases from the end of the exponential growth in K-12 and is variable among natural isolates. Implications of this defective mismatch repair activity for evolution and tumorigenesis will be discussed.     

Production of Autoinducer 2 in Salmonella enterica Serovar Thompson Contributes to Its Fitness in Chickens but Not on Cilantro Leaf Surfaces

Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS⁻ mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS⁻ strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS⁻ mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS⁻ mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.     

Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Km^r as a selectable marker, the insertion element InsTet^G−1 carries the anhydrotetracycline (atc)-regulated outward-directed P_A promoter so that atc-dependent conditional gene knockouts or knockdowns are generated. The complex formed between the purified hyperactive transposase and InsTet^G−1 was electroporated into Escherichia coli or Salmonella enterica serovar Typhimurium, and mutant pools were collected. We used E. coli strains with either TetR or the reverse variant revTetR^r2, while only TetR was employed in Salmonella. Screening of the InsTet^G−1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants for E. coli and 4 atc-regulatable auxotrophic mutants for Salmonella. We have also screened one Salmonella mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTet^G−1 insertion in the gene pyrE or argA survived this procedure, indicating a reduced intracellular growth rate in J774-A.1 cells. The nature of the mutants and the modes of their regulation are discussed.     

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage,despite rapid and sustained mitochondrial DNA depletion,altered energetics and growth

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg^+/− offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg^−/− mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg^+/− and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg^−/− animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg^−/− animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg^−/− mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.     

Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log₁₀/100 cm² of hides and 0.75 log₁₀/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.     

Mutator mutations in Escherichia coli induced by the insertionof phage Mu and the transposable resistance elements Tn5 and Tn10

Mutator mutations in the mutS gene induced by the insertion of phage Mu or the transposable resistance elements Tn5 or Tn10 and those in the mutL gene induced by Tn5 and T10 gave mutagenic activities similar to that of the previously described mutS3 and mutL25 mutations. Various combinations of mutS::Tn5,mutL::Tn5, uvrE156, and the deletion mutation δmutH2 did not produce an additive effect. This supports the idea that the products of these genes function in the same pathway of error correction during DNA synthesis.     

LA35 Poultry Fecal Marker Persistence Is Correlated with That of Indicators and Pathogens in Environmental Waters

Disposal of fecally contaminated poultry litter by land application can deliver pathogens and fecal indicator bacteria (FIB) into receiving waters via runoff. While water quality is regulated by FIB enumeration, FIB testing provides inadequate information about contamination source and health risk. This microbial source tracking (MST) study compared the persistence of the Brevibacterium sp. strain LA35 16S rRNA gene (marker) for poultry litter with that of pathogens and FIB under outdoor, environmentally relevant conditions in freshwater, marine water, and sediments over 7 days. Salmonella enterica, Campylobacter jejuni, Campylobacter coli, Bacteroidales, and LA35 were enumerated by quantitative PCR (qPCR), and Enterococcus spp. and E. coli were quantified by culture and qPCR. Unlike the other bacteria, C. jejuni was not detectable after 48 h. Bacterial levels in the water column consistently declined over time and were highly correlated among species. Survival in sediments ranged from a slow decrease over time to growth, particularly in marine microcosms and for Bacteroidales. S. enterica also grew in marine sediments. Linear decay rates in water (k) ranged from −0.17 day⁻¹ for LA35 to −3.12 day⁻¹ for C. coli. LA35 levels correlated well with those of other bacteria in the water column but not in sediments. These observations suggest that, particularly in the water column, the fate of LA35 in aquatic environments is similar to that of FIB, C. coli, and Salmonella, supporting the hypothesis that the LA35 marker gene can be a useful tool for evaluating the impact of poultry litter on water quality and human health risk.     

The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

The flagellum is a major virulence factor of motile pathogenic bacteria. This structure requires more than 50 proteins for its biogenesis and function, one of which is FlgJ. Homologs of FlgJ produced by the β- and γ-proteobacteria, such as Salmonella enterica, Vibrio spp., and both Sphingomonas sp. and Pseudomonas spp. are bifunctional, possessing an N-terminal domain responsible for proper rod assembly and a C-terminal domain possessing peptidoglycan lytic activity. Despite the amount of research conducted on FlgJ from these and other bacteria over the past 15 years, no biochemical analysis had been conducted on any FlgJ and consequently confusion exists as to whether the enzyme is a peptidoglycan hydrolase or a lytic transglycosylase. In this study, we present the development of a novel assay for glycoside lytic enzymes and its use to provide the first enzymatic characterization of the lytic domain of FlgJ from S. enterica as the model enzyme. Surprisingly, FlgJ functions as neither a muramidase nor a lytic transglycosylases but rather as a β-N-acetylglucosaminidase. As such, FlgJ represents the first autolysin with this activity to be characterized from a Gram-negative bacterium. At its optimal pH of 4.0, the Michaelis-Menten parameters of K_m and k_cat for FlgJ from S. enterica were determined to be 0.64 ± 0.18 mg ml⁻¹ and 0.13 ± 0.016 s⁻¹, respectively, using purified PG as substrate. Its catalytic residues were identified as Glu¹⁸⁴ and Glu²²³.     

Poultry-Associated Salmonella enterica subsp. enterica Serovar 4,12:d:− Reveals High Clonality and a Distinct Pathogenicity Gene Repertoire

A European baseline survey during the years 2005 and 2006 has revealed that the monophasic Salmonella enterica subsp. enterica serovar 4,12:d:− was, with a prevalence of 23.6%, the most frequently isolated serovar in German broiler flocks. In Denmark and the United Kingdom, its serovar prevalences were 15.15% and 2.8%, respectively. Although poultry is a major source of human salmonellosis, serovar 4,12:d:− is rarely isolated in humans (approximately 0.09% per year). Molecular typing studies using pulsed-field gel electrophoresis and DNA microarray analysis show that the serovar is highly clonal and lacks genes with known contributions to pathogenicity. In contrast to other poultry-associated serovars, all strains were susceptible to 17 antimicrobial agents tested and did not encode any resistance determinant. Furthermore, serovar 4,12:d:− lacked the genes involved in galactonate metabolism and in the glycolysis and glyconeogenesis important for energy production in the cells. The conclusion of the study is that serovar 4,12:d:− seems to be primarily adapted to broilers and therefore causes only rare infections in humans.     

Mucoidy,Quorum Sensing,Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Inactivation of Salmonella enterica by UV-C Light Alone and in Combination with Mild Temperatures

The aim of this investigation was to study the efficacy of the combined processes of UV light and mild temperatures for the inactivation of Salmonella enterica subsp. enterica and to explore the mechanism of inactivation. The doses to inactivate the 99.99% (4D) of the initial population ranged from 18.03 (Salmonella enterica serovar Typhimurium STCC 878) to 12.75 J ml⁻¹ (Salmonella enterica serovar Enteritidis ATCC 13076). The pH and water activity of the treatment medium did not change the UV tolerance, but it decreased exponentially by increasing the absorption coefficient. An inactivating synergistic effect was observed by applying simultaneous UV light and heat treatment (UV-H). A less synergistic effect was observed by applying UV light first and heat subsequently. UV did not damage cell envelopes, but the number of injured cells was higher after a UV-H treatment than after heating. The synergistic effect observed by combining simultaneous UV and heat treatment opens the possibility to design combined treatments for pasteurization of liquid food with high UV absorptivity, such as fruit juices.