Assessing genetic diversity in plasmids from Escherichia coli and Salmonella enterica using a mixed-plasmid microarray

AIMS: To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. METHODS AND RESULTS: Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates (r = 0.91) compared with the correlation for nonreplicates (r = 0.09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene (bla(CMY-2)). CONCLUSION: Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. SIGNIFICANCE AND IMPACT OF STUDY: Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.     

Cell-to-cell signalling in Escherichia coli and Salmonella enterica

Cell-to-cell signalling in prokaryotes that leads to co-ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host-microbe interactions. The Gram-negative quorum-sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal-generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal-generating component of a second system (a LuxS homologue that generates AI-2), but the sensory apparatus for AI-2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI-2 in Salmonella encode an active uptake and modification system for AI-2. Therefore, it is not yet clear whether Salmonella uses AI-2 as a signal molecule or whether AI-2 has some other function. In E. coli, the functions of both SdiA and AI-2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Conjugal transfer of natural plasmids between Escherichia coli strains in sterile environmental water

Seven antibiotic-multiresistant Escherichia coli strains, possessing three or four plasmids, capable of transferring their resistance marker at a high frequency, were selected among a total of 300 antibiotic-resistant E. coli strains isolated from natural water—raw and treated wastewater, and brackish water (collected 1 km downstream). These strains were mated with E. coli K-12 C600 nal^r, both in sterilized natural water and LB medium at 25°C. Conjugation did occur in all the systems tested, although fewer transconjugants were recovered from raw and treated wasterwater experiments. In contrast, in brackish and seawater, the transfer frequency did not significantly decrease in spite of salt contents. In 100% of the cases, transfer of the high-molecular-weight plasmids (20 kb) was observed, but the small plasmids (2.6–7.5 kb) were only cotransferred in raw or treated wastewater and in brackish water. Moreover, genotypic variation occurred more frequently in natural water than in LB medium.     

The spread of plasmids in model populations of Escherichia coli K12.

Comparison of R100 with its derepressed derivative R100-1 showed that the capacity to repress tra function does not significantly affect the spread by retransfer of R100. F′lac was used to investigate the contributions of growth and transfer to spread of a plasmid through a recipient population. Ability to transfer F′lac was lost rapidly when donor cultures entered stationary phase, but aggregate-forming ability was lost much more slowly. Comparison of F′lactra⁺ with F′lactraH88, which is unable to retransfer from recipients, showed the importance of retransfer. We used a mathematical model to calculate the amount of retransfer needed to explain the rate of increase of F′lac progeny. This showed that the lag between a cell receiving F′lac and being able to retransfer it was a less important constraint on this rate of increase than the inherent rate of plasmid transfer by established donors.     

Standard reference strains of Escherichia coli from natural populations.

A set of 72 reference strains of Escherichia coli isolated from a variety of hosts and geographical locations has been established for use in studies of variation and genetic structure in natural populations. The strains, which have been characterized by multilocus enzyme electrophoresis, are representative of the range of genotypic variation in the species as a whole.     

Convergent Molecular Evolution of Genomic Cores in Salmonella enterica and Escherichia coli

One of the strongest signals of adaptive molecular evolution of proteins is the occurrence of convergent hot spot mutations: repeated changes in the same amino acid positions. We performed a comparative genome-wide analysis of mutation-driven evolution of core (omnipresent) genes in 17 strains of Salmonella enterica subspecies I and 22 strains of Escherichia coli. More than 20% of core genes in both Salmonella and E. coli accumulated hot spot mutations, with a predominance of identical changes having recent evolutionary origin. There is a significant overlap in the functional categories of the adaptively evolving genes in both species, although mostly via separate molecular mechanisms. As a strong evidence of the link between adaptive mutations and virulence in Salmonella, two human-restricted serovars, Typhi and Paratyphi A, shared the highest number of genes with serovar-specific hot spot mutations. Many of the core genes affected by Typhi/Paratyphi A-specific mutations have known virulence functions. For each species, a list of nonrecombinant core genes (and the hot spot mutations therein) under positive selection is provided.     

No genetic barriers between Salmonella enterica serovar typhimurium and Escherichia coli in SOS-induced mismatch repair-deficient cells

Conjugational crosses trigger SOS induction in Escherichia coli F(-) cells mated with Salmonella enterica serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Solvent accessibility and purifying selection within proteins of Escherichia coli and Salmonella enterica

The neutral theory of molecular evolution predicts that variation within species is inversely related to the strength of purifying selection, but the strength of purifying selection itself must be related to physical constraints imposed by protein folding and function. In this paper, we analyzed five enzymes for which polymorphic sequence variation within Escherichia coli and/or Salmonella enterica was available, along with a protein structure. Single and multivariate logistic regression models are presented that evaluate amino acid size, physicochemical properties, solvent accessibility, and secondary structure as predictors of polymorphism. A model that contains a positive coefficient of association between polymorphism and solvent accessibility and separate intercepts for each secondary-structure element is sufficient to explain the observed variation in polymorphism between sites. The model predicts an increase in the probability of amino acid polymorphism with increasing solvent accessibility for each protein regardless of physicochemical properties, secondary-structure element, or size of the amino acid. This result, when compared with the distribution of synonymous polymorphism, which shows no association with solvent accessibility, suggests a strong decrease in purifying selection with increasing solvent accessibility.     

Recombination and clonality in natural populations of Escherichia coli

Bacteria such as Escherichia coli have been commonly viewed as being primarily clonal organisms. As such, the genetic variation within clones was thought to be almost exclusively the result of the mutational process. This conclusion has recently been challenged by data from DNA sequencing studies of natural isolates that are incompatible with a primarily clonal structure. Molecular population genetic analyses of these data, including gene genealogical comparisons, have raised the possibility of a much more complex population structure that may encompass relatively frequent recombination, recurrent selective sweeps and extensive ecological population subdivision.     

Evolution of Pan-Genomes of Escherichia coli,Shigella spp., and Salmonella enterica

Multiple sequencing of genomes belonging to a bacterial species allows one to analyze and compare statistics and dynamics of the gene complements of species, their pan-genomes. Here, we analyzed multiple genomes of Escherichia coli, Shigella spp., and Salmonella enterica. We demonstrate that the distribution of the number of genomes harboring a gene is well approximated by a sum of two power functions, describing frequent genes (present in many strains) and rare genes (present in few strains). The virtual absence of Shigella-specific genes not present in E. coli genomes confirms previous observations that Shigella is not an independent genus. While the pan-genome size is increasing with each new strain, the number of genes present in a fixed fraction of strains stabilizes quickly. For instance, slightly fewer than 4,000 genes are present in at least half of any group of E. coli genomes. Comparison of S. enterica and E. coli pan-genomes revealed the existence of a common periphery, that is, genes present in some but not all strains of both species. Analysis of phylogenetic trees demonstrates that rare genes from the periphery likely evolve under horizontal transfer, whereas frequent periphery genes may have been inherited from the periphery genome of the common ancestor.     

Acquisition of virulence-associated factors by the enteric pathogens Escherichia coli and Salmonella enterica

In this review we summarize recent genomic studies that shed light on the mechanism through which pathogenic Escherichia coli and Salmonella enterica have evolved. We show how acquisition of DNA at specific sites on the chromosome has contributed to increased genetic variation and virulence of these two genera of the Enterobacteriaceae.     

YhdJ, a nonessential CcrM-like DNA methyltransferase of Escherichia coli and Salmonella enterica

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the alpha-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.     

Homology between Escherichia coli plasmids ColE1 and p15A.

The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented.     

The beta-oxidation systems of Escherichia coli and Salmonella enterica are not functionally equivalent

Based on its genome sequence, the pathway of beta-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to grow, whereas S. enterica grew well. Exchange of the wild-type fadR genes between the two organisms showed this to be a property of S. enterica rather than of the FadR proteins per se. This difference in growth was attributed to S. enterica having higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid. S. enterica cleanly converted all of the acid to acetyl-CoA, whereas E. coli accumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that the S. enterica FadE and FadBA enzymes were responsible for the greater efficiency of beta-oxidation relative to that of E. coli.     

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed.     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.