Dvora Biran

Host Imprints on Bacterial Genomes-Rapid, Divergent Evolution in Individual Patients

Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strains evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.

Regulated proteolysis in Gram-negative bacteria — how and when?

Most bacteria live in a dynamic environment where temperature, availability of nutrients and the presence of various chemicals vary, which requires rapid adaptation. Many of the adaptive changes are determined by changes in the transcription of global regulatory networks, but this response is slow because most bacterial proteins are stable and their concentration remains high even after transcription slows down. To respond rapidly, an additional level of regulation has evolved: the degradation of key proteins. However, as proteolysis is an irreversible process, it is subject to tight regulation of substrate binding and degradation. Here we review the roles of the proteolytic enzymes in Gram-negative bacteria and how these enzymes can be regulated to target only a subset of proteins.

Control of methionine biosynthesis in Escherichia coli by proteolysis

Most bacterial proteins are stable, with half‐lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis – homoserine trans‐succinylase (HTS) – is unstable and subject to energy‐dependent proteolysis. The enzyme is stable in triple mutants defective in Lon‐, HslVU‐ and ClpP‐dependent proteases. The instability of the protein is determined by the amino‐terminal part of the protein, and its removal or substitution by the N‐terminal part of β‐galactosidase confers stability. The effect of the amino‐terminal segment is not caused by the N‐end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half‐life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.

In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures

The formation of protein aggregates is associated with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temperatures in minimal media. Homoserine trans‐succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44°C in vitro. Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non‐permissive temperatures. This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild‐type bacteria.

Transfer of noncoding DNA drives regulatory rewiring in bacteria

Significance The rapid pace of evolution in bacteria is widely attributed to the promiscuous horizontal transfer and recombination of protein-coding genes. However, it has not been investigated if the same forces also drive the evolution of noncoding regulatory regions. Here, we establish that regulatory regions can “switch” between nonhomologous alternatives and that switching is ubiquitous, occurring across the bacterial domain. We show that regulatory switching has a strong impact on promoter architecture and expression divergence. Further, we demonstrate that regulatory transfer facilitates rapid phenotypic diversification of a human pathogen. This regulatory mobility enables bacterial genes to access a vast pool of potential regulatory elements, facilitating efficient exploration of the regulatory landscape. Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory “switching” to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.

Probing the active site of homoserine trans-succinylase

Homoserine trans‐succinylase is the first enzyme in methionine biosynthesis of Escherichia coli and catalyzes the activation of homoserine via a succinylation reaction. The in vivo activity of this enzyme is subject to tight regulation by several mechanisms, including repression and activation of gene expression, feedback inhibition, temperature regulation and proteolysis. This complex regulation reflects the key role of this enzyme in bacterial metabolism. Here, we demonstrate – using proteomics and high‐resolution mass spectrometry – that succinyl is covalently bound to one of the two adjacent lysine residues at positions 45 and 46. Replacing these lysine residues by alanine abolished the enzymatic activity. These findings position the lysine residues, one of which is conserved, at the active site.

Adaptation of Escherichi coli to elevated temperatures involves a change in stability of heat shock gene transcripts

Bacteria respond to shift-up in temperature by activating the heat shock response - induction of a large number of heat shock genes. This response is essential for adaptation to the higher temperature and for acquiring thermotolerance. One unique feature of the heat shock response is its transient nature - shortly after the induction, the rate of synthesis of heat shock proteins decreases, even if the temperature remains high. Here we show that this shutoff is due to a decrease in the transcript stability of heat shock genes. We further show that the modulation of stability of mRNAs of heat shock genes is maintained by the cold shock protein C - CspC - previously shown to affect transcript stability of specific genes. Upon shifts to higher temperatures the level of this protein decreases due to proteolysis and aggregation, leading to a reduced stability of mRNAs of heat shock genes. The temperature-dependent modulation of transcript stability of heat shock genes constitutes a novel control of the bacterial response to temperature changes.

The Escherichia coli DjlA and CbpA Proteins Can Substitute for DnaJ in DnaK-Mediated Protein Disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.

Proteome analysis of plant-induced proteins of Agrobacterium tumefaciens

Abstract A proteome study of Agrobacterium tumefaciens exposed to plant roots demonstrated the existence of a plant-dependent stimulon. This stimulon was induced by exposure to cut roots and consists of at least 30 soluble proteins (pI 4-7), including several proteins whose involvement in agrobacteria-host interactions has not been previously reported. Exposure of the bacteria to tomato roots also resulted in modification of the proteins: Ribosomal Protein L19, GroEL, AttM, and ChvE, indicating the significance of protein modifications in the interactions of agrobacteria with plants.

Genomic Avenue to Avian Colisepticemia

ABSTRACT Here we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenic Escherichia coli (APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. coli type 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of the Yersinia high-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum. IMPORTANCE Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease. Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.