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PubMed2006

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase.

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.

Anti-Bacterial Agents↗
PubMed2001

Protected environments allow parallel evolution of a bacterial pathogen in a patient subjected to long-term antibiotic therapy.

Long-term antibiotic treatment offers a rare opportunity to study the evolution of bacteria within the same individual. The appearance of new variants has been suggested to take place via the selection of enhanced resistance in compartments of the body in which the antibiotic concentration is low. Laboratory models of protected compartments have elegantly demonstrated their potential in selecting novel variants. However, comparable data from patients have been rare. In this study, extended antibiotic therapy in a single patient suffering from multiple infected liver cysts has provided the opportunity to observe and analyse the molecular evolution of antibiotic resistance. Each isolate has the same basic ompC gene sequence that is distinct from other Escherichia coli isolates, which suggests that they derive from the same founder population. However, the isolates differ in their auxotrophic markers, in the pI values of their dominant beta-lactamase activities and in the mutations in the promoter region of the ampC gene leading to increased expression of the AmpC enzyme. The data provide strong evidence for a single focal infection expanding via parallel pathways of evolution to give a range of antibiotic-resistant isolates. These data suggest that the infected cysts provide numerous protected environments that are the foci for the separate development of distinct variants.

Adult↗
PubMed1995

Tracking the evolution of the bacterial choline-binding domain: molecular characterization of the Clostridium acetobutylicum NCIB 8052 cspA gene.

The major secreted protein of Clostridium acetobutylicum NCIB 8052, a choline-containing strain, is CspA (clostridial secreted protein). It appears to be a 115,000-M(r) glycoprotein that specifically recognizes the choline residues of the cell wall. Polyclonal antibodies raised against CspA detected the presence of the protein in the cell envelope and in the culture medium. The soluble CspA protein has been purified, and an oligonucleotide probe, prepared from the determined N-terminal sequence, has been used to clone the cspA gene which encodes a protein with 590 amino acids and an M(r) of 63,740. According to the predicted amino acid sequence, CspA is synthesized with an N-terminal segment of 26 amino acids characteristic of prokaryotic signal peptides. Expression of the cspA gene in Escherichia coli led to the production of a major anti-CspA-labeled protein of 80,000 Da which was purified by affinity chromatography on DEAE-cellulose. A comparison of CspA with other proteins in the EMBL database revealed that the C-terminal half of CspA is homologous to the choline-binding domains of the major pneumococcal autolysin (LytA amidase), the pneumococcal antigen PspA, and other cell wall-lytic enzymes of pneumococcal phages. This region, which is constructed of four repeating motifs, also displays a high similarity with the glucan-binding domains of several streptococcal glycosyltransferases and the toxins of Clostridium difficile.

Amino Acid Sequence↗
PubMed2003

Optical processing of bacterial libraries for directed evolution.

Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer-controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56-fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries.

Cell Division↗
PubMed2001

Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins).

In the gram-negative model organism Escherichia coli, the effector molecule of the stringent response, (p)ppGpp, is synthesized by two different enzymes, RelA and SpoT, whereas in the gram-positive model organism Bacillus subtilis only one enzyme named Rel is responsible for this activity. Rel and SpoT also possess (p)ppGpp hydrolase activity. BLAST searches were used to identify orthologous genes in databases. The construction and bootstrapping of phylogenetic trees allowed classification of these orthologs. Four groups could be distinguished: With the exception of Neisseria and Bordetella (beta subdivision), the RelA and SpoT groups are exclusively found in the gamma subdivision of proteobacteria. Two Rel groups representing the actinobacterial and the Bacillus/Clostridium group were also identified. The SpoT proteins are related to the gram positive Rel proteins. RelA proteins carry substitutions in the HD domain (Aravind and Koonin, 1998, TIBS 23: 469-472) responsible for ppGpp degradation. A theory for the evolution of the specialized, paralogous relA and spoT genes is presented: After gene duplication of an ancestral rellike gene, the spoT and relA genes evolved from the duplicated genes. The distribution pattern of the paralogous RelA and SpoT proteins supports a new model of linear bacterial evolution (Gupta, 2000, FEMS Microbiol. Rev. 24: 367-402). This model postulates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. However, two paralogous, closely related genes of Porphyromonas gingivalis (Cytophaga-Flavobacterium-Bacteroides phylum) encoding proteins with functions probably identical to the RelA and SpoT proteins do not fit in this model. Completely sequenced genomes of several obligately parasitic organisms (Treponema pallidum, Chlamydia species, Rickettsia prowazekii) and the obligate aphid symbiont Buchnera sp. APS as well as archaea do not contain rel-like genes but they are present in the Arabidopsis genome. In crosslinking experiments using different analogs of ppGpp as crosslinking reagents and RNA polymerase preparations of Escherichia coli, binding of ppGpp to distinct regions at the C-terminus of the beta subunit (the RpoB gene product) and/or at the N-terminus of the beta subunit (the RpoC gene product) was observed previously. RpoB and RpoC sequences of the species which do not possess a rel like gene do not exhibit specific insertions or deletions in the ppGpp binding regions.

Amino Acid Substitution↗
PubMedDate not supplied

Bacterial active-site serine penicillin-interactive proteins and domains: mechanism, structure, and evolution.

The bacterial active-site serine penicillin-hydrolyzing and penicillin-binding proteins or domains operate by a serine-ester-linked acyl enzyme mechanism similar to that of the peptidases of the trypsin and subtilisin families. On the basis of known primary and tertiary structures, predictive studies support the view that these proteins or domains form a superfamily of evolutionarily related enzymes. Although--depending on the evolutionary distance--they may have very different amino acid sequences and distinct functional characteristics and specificities, they all would have conserved the same unprecedented type of polypeptide scaffolding. When obtained, complete structural information should provide the necessary tools for the rational design of novel types of inactivators of these important enzyme targets.

Acyltransferases↗
PubMed2006

Bacterial genomics and pathogen evolution.

The availability of hundreds of bacterial genome sequences has altered the study of bacterial pathogenesis, affecting both design of experiments and analysis of results. Comparative genomics and genomic tools have been used to identify virulence factors and genes involved in environmental persistence of pathogens. However, a major stumbling block in the genomics revolution has been the large number of genes with unknown function that have been identified in every organism sequenced to date.

Bacteria↗
PubMed2005

Evolution of the bacterial phosphotransferase system: from carriers and enzymes to group translocators.

The bacterial phosphotransferase system (PTS) is a structurally and functionally complex system with a surprising evolutionary history. The substrate-recognizing protein constituents of the PTS (Enzymes II) derive from at least four independent sources. Some of the non-PTS precursor constituents have been identified, and evolutionary pathways taken have been proposed. Our analyses suggest that two of these independently evolving systems are still in transition, not yet having acquired the full-fledged characteristics of PTS Enzyme II complexes. The work described provides detailed insight into the process of catalytic protein evolution.

Bacteria↗
PubMed1990

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes.

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Helicobacter pylori: molecular evolution of a bacterial quasi-species.

Helicobacter pylori persists chronically within individuals and as they spread the mutating bacteria migrate with them. The continuous selection and microevolution generates a population of closely related but different bacteria that behave like a quasi-species. Within this heterogeneity, H. pylori strains fall into distinct types, into the virulent (type I) and less virulent (type II) strains, based on the presence of a pathogenicity island (cag) that encodes a specialized secretion machinery. We propose that during chronic infection a dynamic equilibrium between bacteria expressing a disparate degree of virulence is established, and that diverse forms prevail at different times.

Animals↗
PubMed1988

Organization and evolution of the bacterial genome.

The bacterial chromosome seems to be organized in such a way as to allow the formation of tandem gene duplications, which appear under conditions of positive selection for a high level of gene expression. Multiple gene duplications permit the excision of a gene from the chromosome by a Campbell recombinational event and the subsequent transfer of the gene to a resident plasmid. The latter derives an advantage from carrying a gene providing positive selection. Thus, tandem gene duplications could initiate horizontal gene transfer in bacteria.

Bacteria↗
PubMed1999

RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria.

A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.

Adenosine Triphosphatases↗
PubMed1986

[Construction of the bacterial genome--the next goal of genetic engineering].

The article deals primarily with literature data on the organization and evolution of bacterial genome. An account of the conception of "current" bacterial evolution consisting of periodical inclusions into a bacterial genome of genes having exogenous origin is given. A hypothesis is proposed that rare inversions going through the oriC or terC chromosomal regions could serve as reparative rearrangements providing balanced distribution of the DNA segments of different origin within the circular genome. It is asserted that the progress in the study of the model microorganism Escherichia coli K-12 as well as in genetical methods give rise to a new experimental challenge--the creation of a "rational" bacterial genome on the basis of E. coli cell. It is proposed that a "rational" bacterial genome could be devoid of many genes which provide the evolutionary formed ecological potential of the species and its capacity for rapid adaptation to new environment.

Biological Evolution↗
PubMed2005

Enterohemorrhagic Escherichia coli in human infection: in vivo evolution of a bacterial pathogen.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) cause most cases of the hemolytic uremic syndrome (HUS) worldwide. To investigate genetic changes in EHEC during the course of human infection, we analyzed consecutive stool samples and shed isolates from patients with HUS, focusing on the genes encoding Shiga toxin (stx) and intimin (eae). METHODS: Sequential stool samples from 210 patients with HUS were investigated for the persistence of E. coli strains harboring stx and/or eae. Initial stool samples were collected during the acute phase of HUS, and subsequent stool samples were collected 3-16 days later (median interval, 8 days). RESULTS: Organisms that were stx and eae positive (stx+/eae+ strains; n=137) or stx negative and eae positive (stx-/eae+ strains; n=5) were detected in the initial stool samples from 142 patients. Subsequently, the proportion of those who shed stx+/eae+ strains decreased to 13 of 210 patients, whereas the proportion of those who shed strains that were stx-/eae+ increased to 12 of 210 patients. Seven patients who initially excreted strains that were stx+/eae+ shed, at second analysis, stx-/eae+ strains of the same serotypes; they had no free fecal Shiga toxin at follow-up. Comparison of the initial and follow-up isolates from these patients with use of molecular-epidemiological methods revealed loss of stx genes and genomic rearrangement. CONCLUSIONS: We demonstrate the loss of a critical bacterial virulence factor from pathogens during very brief intervals in the human host. These genetic changes have evolutionary, diagnostic, and clinical implications. Generation of stx- mutants might contribute to subclonal evolution and evolutionary success.

Enterotoxins↗
PubMed2003

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity.

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Amino Acid Sequence↗