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Steffen Porwollik

Publications and source records attributed to Steffen Porwollik.

At least 19 recordsLinked to original sources
PubMed2026

Identification of genes promoting fitness of a plant-associated Salmonella Choleraesuis strain on alfalfa sprouts during cold storage.

Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their nutritional value and antioxidant content. However, these products have repeatedly been implicated in outbreaks of foodborne pathogens, including Salmonella enterica. Although host-adapted Salmonella serovars are less frequently associated with foodborne illness, infections caused by these serovars often result in invasive and severe outcomes, highlighting the importance of understanding their persistence in food production systems. Moreover, the variability among Salmonella serovars requires characterization beyond the most prevalent types to support the development of precision food safety strategies effective across the diversity of serovars capable of contaminating fresh produce. Here, a plant-internalized Salmonella Choleraesuis strain was used as a model to investigate persistence mechanisms on alfalfa sprouts. A bar-coded transposon mutant library comprising approximately 33,000 unique insertions was generated, along with a collection of individual insertion mutants. These resources were used to identify genetic determinants contributing to strain fitness on sprouts under abusive cold storage (8°C) simulating commercial shelf-life environments. Genome-wide analyses identified negative selection for mutants with insertions in eda, fabF, lpp1_2, pnp, stpA, SCHChr_03621, and two intergenic regions. Competition assays confirmed fitness defects associated with eda, encoding a key enzyme of the Entner-Doudoroff pathway; mnmG, encoding a tRNA modification enzyme involved in translational fidelity; and fabF, involved in fatty acid biogenesis. These findings provide a genome-wide perspective on mechanisms enabling persistence on sprouts of a plant-associated, host-adapted Salmonella strain during cold storage and inform risk assessment and intervention design within precision food safety frameworks.IMPORTANCEFood safety strategies are frequently based on knowledge derived from well-studied, epidemiologically relevant Salmonella serovars, yet many less frequent types still pose a risk to consumers and may contaminate fresh produce. Different Salmonella serovars may vary in the relative contribution of persistence mechanisms. Recognizing these differences is essential for improving precision food safety efforts, particularly for foods such as sprouts that are repeatedly linked to outbreaks. This study highlights that less-studied serovars can rely on both shared survival strategies and unique traits that might otherwise not be captured by current control approaches. By demonstrating that strain diversity influences persistence on fresh produce, this work supports the development of precision food safety strategies that address a broader spectrum of Salmonella, thereby improving risk assessment and helping to better protect public health.

food safety↗
PubMed2007

FNR is a global regulator of virulence and anaerobic metabolism in Salmonella enterica serovar Typhimurium (ATCC 14028s).

Salmonella enterica serovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. In Escherichia coli, FNR is one of the main regulatory proteins involved in O2 sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in serovar Typhimurium is similar to, but distinct from, that in E. coli. Thus, genes/operons involved in aerobic metabolism, NO. detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a fashion similar to that in E. coli. However, genes/operons existing in E. coli but regulated by FNR only in serovar Typhimurium include those coding for ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly, Salmonella-specific genes/operons regulated by FNR include numerous virulence genes within Salmonella pathogenicity island 1 (SPI-1), newly identified flagellar genes (mcpAC, cheV), and the virulence operon (srfABC). Furthermore, the role of FNR as a positive regulator of motility, flagellar biosynthesis, and pathogenesis was confirmed by showing that the mutant is nonmotile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

Anaerobiosis↗
PubMed2006

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica.

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.

Animals↗
PubMed2006

Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella.

Horizontal gene transfer plays a major role in microbial evolution. However, newly acquired sequences can decrease fitness unless integrated into preexisting regulatory networks. We found that the histone-like nucleoid structuring protein (H-NS) selectively silences horizontally acquired genes by targeting sequences with GC content lower than the resident genome. Mutations in hns are lethal in Salmonella unless accompanied by compensatory mutations in other regulatory loci. Thus, H-NS provides a previously unrecognized mechanism of bacterial defense against foreign DNA, enabling the acquisition of DNA from exogenous sources while avoiding detrimental consequences from unregulated expression of newly acquired genes. Characteristic GC/AT ratios of bacterial genomes may facilitate discrimination between a cell's own DNA and foreign DNA.

Bacterial Proteins↗
PubMed2006

Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences.

The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1- and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial peptides, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.

Cell Line↗
PubMed2006

DNA microarray detection of antimicrobial resistance genes in diverse bacteria.

High throughput genotyping is essential for studying the spread of multiple antimicrobial resistance. A test oligonucleotide microarray designed to detect 94 antimicrobial resistance genes was constructed and successfully used to identify antimicrobial resistance genes in control strains. The microarray was then used to assay 51 distantly related bacteria, including Gram-negative and Gram-positive isolates, resulting in the identification of 61 different antimicrobial resistance genes in these bacteria. These results were consistent with their known gene content and resistance phenotypes. Microarray results were confirmed by polymerase chain reaction and Southern blot analysis. These results demonstrate that this approach could be used to construct a microarray to detect all sequenced antimicrobial resistance genes in nearly all bacteria.

Bacteria↗
PubMed2006

DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens?

Genomic hybridization on whole genome arrays detects the presence or absence of similar DNA regions in sufficiently related microorganisms, allowing genome-wide comparison of their genetic contents. A whole genome array is based on a sequenced bacterial isolate, and is a collection of DNA probes fixed on a solid support. In a single hybridization experiment, the absence/presence status of all genes of the sequenced microbe in the queried isolate can be examined. The objective of this minireview is to summarize the past usage of DNA microarray technology for microbial strain characterizations, and to estimate its future utilization in epidemiological studies and molecular typing of bacterial pathogens. The studies reviewed here confirm the usefulness of microarray technology for the detection of genetic polymorphisms. However, the construction or purchase of DNA microarrays and the performance of strain to strain hybridization experiments are still prohibitively expensive for routine application. Future use of arrays in epidemiology is likely to depend on the development of more cost-effective protocols, more robust and simplified formats, and the adequate evaluation of their performance (efficacy) and convenience (efficiency) compared with other genotyping methods. It seems more likely that a more focused assay, concentrating on genomic regions of variability previously detected by genome-wide microarrays, will find broad application in routine bacterial epidemiology.

Bacteria↗
PubMed2005

Sensing wetness: a new role for the bacterial flagellum.

We have uncovered a new role for the bacterial flagellum in sensing external wetness. An investigation into why mutants in the chemotaxis signaling pathway of Salmonella typhimurium exhibit fewer and shorter flagella than wild-type when propagated on a surface, first showed that the mutants downregulate only a small set of genes on swarm media--class 3 or 'late' motility genes, and genes associated with the pathogenicity island SPI-1 TTSS (type three secretion system). Based on observations that swarm colonies of the mutants appear less hydrated, we tested a model in which the flagellum itself is a sensor: suboptimal external hydration interferes with secretion of flagellin subunits, inhibiting filament growth and blocking normal export of the class 3 transcription inhibitor FlgM. We provide strong experimental support for the model. In addition, the data show that the flagellar and SPI-1 TTSS are coupled via regulatory proteins. These studies implicate the flagellum, a bacterial organ for motility, in sensing the external environment to modulate not only its own biogenesis but other physiological functions as well.

Bacterial Proteins↗
PubMed2005

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis.

The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37 degrees C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25 degrees C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25 degrees C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37 degrees C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.

Animals↗
PubMed2005

Host gene expression changes and DNA amplification during temperate phage induction.

Salmonella enterica serovar Typhimurium LT2 harbors four temperate prophages. The lytic cycle of these phages was induced with hydrogen peroxide or mitomycin C. Microarray analysis was used to monitor the increase in phage genome copy number and the changes in RNA expression. Phage gene transcription was classified temporally, and host genes that responded to hydrogen peroxide, mitomycin C, or phage induction were also identified. A region of the serovar Typhimurium LT2 host genome encompassing hundreds of genes, flanking the Fels-1 lambdoid prophage, was amplified manyfold during lytic induction, presumably due to Fels-1 runoff replication prior to excision, a phenomenon termed escape replication. An excisionase (xis) mutant of Fels-1 also induced escape replication but did not get packaged. Gifsy-1, a lambdoid prophage that does not normally produce escape replication, did so after deletion of either its integrase or excisionase genes. Escape replication is probably widespread; large regions of host genome amplification were also observed after phage induction in serovar Typhimurium strains SL1344 and 14028s at the suspected integration site of prophage genomes.

DNA Nucleotidyltransferases↗
PubMed2004

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Base Sequence↗
PubMed2004

DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures.

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.

Biological Specimen Banks↗
PubMed2004

Host restriction of Salmonella enterica serotype Typhimurium pigeon isolates does not correlate with loss of discrete genes.

The definitive phage types (DT) 2 and 99 of Salmonella enterica serotype Typhimurium are epidemiologically correlated with a host range restricted to pigeons, in contrast to phage types with broader host ranges such as epidemic cattle isolates (DT104 and DT204). To determine whether phage types with broad host range possess genetic islands absent from host-restricted phage types, we compared the genomes of four pigeon isolates to serotype Typhimurium strain LT2 using a DNA microarray. Three of the four isolates tested caused fluid accumulation in bovine ligated ileal loops, but they had reduced colonization of liver and spleen in susceptible BALB/c mice and were defective for intestinal persistence in Salmonella-resistant CBA mice. The genomes of the DT99 and DT2 isolates were extremely similar to the LT2 genome, with few notable differences on the level of complete individual genes. Two large groups of genes representing the Fels-1 and Fels-2 prophages were missing from the DT2 and DT99 phage types we analyzed. One of the DT99 isolates examined was lacking a third cluster of five chromosomal genes (STM1555 to -1559). Results of the microarray analysis were extended using Southern analysis to a collection of 75 serotype Typhimurium clinical isolates of 24 different phage types. This analysis revealed no correlation between the presence of Fels-1, Fels-2, or STM1555 to -1559 and the association of phage types with different host reservoirs. We conclude that serotype Typhimurium phage types with broad host range do not possess genetic islands influencing host restriction, which are absent from the host-restricted pigeon isolates.

Animals↗
PubMed2003

A non-redundant microarray of genes for two related bacteria.

A microarray with sequences from the annotated open reading frames (ORFs) in Salmonella enterica subspecies 1, serovar Typhimurium was supplemented with annotated chromosomal ORFs from serovar Typhi that are divergent from Typhimurium (>10% DNA sequence divergence). This non- redundant array was used to (i) measure changes in gene copy number in DNA from actively growing versus stationary Typhi and (ii) to reveal the transcriptional response of Typhi to peroxide, a stress similar to that experienced when they are phagocytosed by macrophages. In S.enterica subspecies 1, pairs of genomes differ in the presence or absence of approximately 10% of their genes. An array twice the size of that needed to cover all ORFs for one genome could carry close homologs of all the ORFs for 10 genomes. Non-redundant DNA arrays could be constructed for any group of closely related organisms that differ by the presence and absence of a few genes.

Gene Expression Profiling↗
PubMed2003

Lateral gene transfer in Salmonella.

Comparative genomics and microarrays reveal that the genomes of different Salmonella enterica serovars are distinguished from each other by the presence or absence of hundreds of genes. The distribution of these variable genome regions is often not clonal. Therefore, lateral gene transfer (LGT) plays an important role in diversity among Salmonella. Overall, almost one quarter of the entire S. enterica sv Typhimurium genome may have been introduced by LGT.

Gene Transfer, Horizontal↗
PubMed2003

Global regulation by CsrA in Salmonella typhimurium.

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Animals↗