Barry L. Wanner

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

We have systematically made a set of precisely defined, single‐gene deletions of all nonessential genes in Escherichia coli K‐12. Open‐reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one‐step method for inactivation of chromosomal genes and primers designed to create in‐frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high‐throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E. coli K‐12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist‐nara.ac.jp/).

Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.

Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.

Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12

We have systematically examined the mRNA profiles of 36 two‐component deletion mutants, which include all two‐component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria–Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two‐component systems. A high correlation coefficient of expression profile was found between several two‐component mutants. Together, these results support the view that a network of functional interactions, such as cross‐regulation, exists between different two‐component systems. The compiled data are avail‐able at our website (http:ecoli.aist‐nara.ac.jpxpanalysis 2components).

Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110

With the goal of solving the whole‐cell problem with Escherichia coli K‐12 as a model cell, highly accurate genomes were determined for two closely related K‐12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single‐nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino‐acid residue deletion, five amino‐acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13 000 bases) were mostly within portions sequenced with out‐dated technology based on radioactive chemistry.

Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) is a small RNA-based adaptive prokaryotic immunity system that functions by acquisition of short fragments of DNA (mainly from foreign invaders such as viruses and plasmids) and subsequent destruction of DNA with sequences matching acquired fragments. Some mutations in foreign DNA that affect the match prevent CRISPR/Cas defensive function. Here we show that matching sequences that are no longer able to elicit defense, still guide the CRISPR/Cas acquisition machinery to foreign DNA, thus making the spacer acquisition process adaptive and leading to restoration of CRISPR/Cas-mediated protection. We present evidence suggesting that after initial recognition of partially matching foreign DNA, the CRISPR/Cas acquisition machinery moves along the DNA molecule, occasionally selecting fragments to be incorporated into the CRISPR locus. Our results explain how adaptive CRISPR/Cas immunity becomes specifically directed towards foreign DNA, allowing bacteria to efficiently counter individual viral mutants that avoid CRISPR/Cas defense.

Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems

Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.

Homogeneous expression of the P BAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter

Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD). In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Multiple Factors Independently Regulate hilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimuriums ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.