High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Quantitative Polymerase Chain Reaction for Microbial Growth Kinetics of Mixed Culture System.

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (µmax and Ks). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

Bacteriophage vB_EcoM_FV3: a new member of "rV5-like viruses".

A proposed new genus of the family Myoviridae, "rV5-like viruses", includes two lytic bacteriophages: Escherichia coli O157: H7-specific bacteriophage rV5 and Salmonella phage PVP-SE1. Here, we present basic properties and genomic characterization of a novel rV5-like phage, vB_EcoM_FV3, which infects E. coli K-12-derived laboratory strains and replicates at high temperature (up to 47 °C). The 136,947-bp genome of vB_EcoM_FV3 contains 218 open reading frames and encodes 5 tRNAs. The genomic content and organization of vB_EcoM_FV3 is more similar to that of rV5 than to PVP-SE1, but all three phages share similar morphological characteristics and form a homogeneous phage group.

The WalKR system controls major staphylococcal virulence genes and is involved in triggering the host inflammatory response.

The WalKR two-component system is essential for the viability of Staphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR in S. aureus through a phosphomimetic amino acid replacement (WalR(c), D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb, emp, fnbA, and fnbB), cytolysis (hlgACB, hla, and hlb), and innate immune defense evasion (scn, chp, and sbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalR(c) are strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively active walR(c) allele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

Outsider to insider: resetting the natural host niche of commensal E. coli K-12.

The status of E. coli K-12 as an exclusively non-invasive, non-pathogenic bacterium has almost been incontrovertible. Our recent finding that a mutation in one of its main architectural protein, HU, converts E. coli K-12 to an actively invasive form suggests that gaining host cell entry might be an expedient survival tactic for traditional commensals during certain altered host conditions. The mutant E. coli (SK3842) exhibits properties usually associated with pathogenic bacteria: host cell invasion, phagosomal disruption and intracellular replication. However, unlike the situation with some pathogens, internalized SK3842 imparts anti-apoptotic and cyto-protective effects rather than lethality on the host cell, both in vitro and in vivo. Here, we show that SK3842 also provides colonization resistance against other invasive pathogens--a trait not shared by the parental commensal strain. Thus, the altered lifestyle of SK3842 encompasses characteristics both from traditional pathogens as well as beneficial probiotic strains.

Newly identified genetic variations in common Escherichia coli MG1655 stock cultures.

We have recently identified seven mutations in commonly used stocks of the sequenced Escherichia coli strain MG1655 which do not appear in the reference sequence. The mutations are likely to cause loss of function of the glpR and crl genes, which may have serious implications for physiological experiments using the affected strains.

Simple quantification of bacterial envelope-associated extracellular materials.

We have developed a simple method for distinguishing between bacterial cultures that produce different amount of exopolysaccharide. It is based upon small differences in pellet volume formed by those cultures upon centrifugation. For that we have constructed a special centrifugation tube consisting of two connected chambers: an upper 12 ml chamber connected to a lower capillary chamber. Cells are applied to the upper chamber and following centrifugation, sink to its bottom and are forced into the capillary so that the height they fill can be measured. This procedure has been developed in order to demonstrate differences in volume of centrifugation pellet formed by similar number of Escherichia coli K12 wild type, rpoS mutant and yjbG rpoS double mutant cells. These differences are further shown to be a result of overproduction of colanic acid exopolysaccharide in the mutant strains. We suggest that this simple method can be employed to detect differences in other cell surface structures and to estimate biomass when optical density measurement or microscopic count is not applicable.

Adaptive evolution in single species bacterial biofilms.

Little is known about the dynamics of cellular growth, death, and evolution within bacterial biofilms. Here we show evidence of evolution within single-species biofilms in real time. Escherichia coli harvested from 22-day-old biofilms express a competitive advantage over cells incubated in biofilms for shorter periods of time. This advantage is manifested as the ability of aged cells to outcompete younger cells in the presence of a pre-existing biofilm, even though cells from older biofilms do not express an increased ability to form initial biofilms on a fresh, unoccupied surface. This phenomenon is similar to the growth advantage in stationary phase, or GASP, phenotype exhibited by planktonically grown cells when incubated under competitive conditions. The ability of bacteria in biofilms to show rapid heritable change has implications for our understanding of the adaptive abilities of biofilms in a wide variety of natural and man-made environments.

Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12.

BACKGROUND: Repairing DNA damage begins with its detection and is often followed by elicitation of a cellular response. In E. coli, RecA polymerizes on ssDNA produced after DNA damage and induces the SOS Response. The RecA-DNA filament is an allosteric effector of LexA auto-proteolysis. LexA is the repressor of the SOS Response. Not all RecA-DNA filaments, however, lead to an SOS Response. Certain recA mutants express the SOS Response (recA(C)) in the absence of external DNA damage in log phase cells. METHODOLOGY/PRINCIPAL FINDINGS: Genetic analysis of two recA(C) mutants was used to determine the mechanism of constitutive SOS (SOS(C)) expression in a population of log phase cells using fluorescence of single cells carrying an SOS reporter system (sulAp-gfp). SOS(C) expression in recA4142 mutants was dependent on its initial level of transcription, recBCD, recFOR, recX, dinI, xthA and the type of medium in which the cells were grown. SOS(C) expression in recA730 mutants was affected by none of the mutations or conditions tested above. CONCLUSIONS/SIGNIFICANCE: It is concluded that not all recA(C) alleles cause SOS(C) expression by the same mechanism. It is hypothesized that RecA4142 is loaded on to a double-strand end of DNA and that the RecA filament is stabilized by the presence of DinI and destabilized by RecX. RecFOR regulate the activity of RecX to destabilize the RecA filament. RecA730 causes SOS(C) expression by binding to ssDNA in a mechanism yet to be determined.

Statistical analyses: possible reasons for unreliability of source tracking efforts.

Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological quality of water include Escherichia coli, a thermotolerant coliform found in the feces of warm-blooded animals. These bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain forests in the absence of fecal contamination. In the present study, E. coli isolates were compared to E. coli K12 (ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically, genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for almost every one of the E. coli isolates analyzed. Cluster analysis did not show any relations between isolates and their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in the classification results; therefore, the large genetic heterogeneity observed in these E. coli populations makes the grouping of isolates by source rather difficult, if not impossible.

Phenotypic screening of Escherichia coli K-12 Tn5 insertion libraries, using whole-genome oligonucleotide microarrays.

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.