The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.     

Site-directed mutagenesis of conserved Thr407, Asp433 and Met464 residues in small subunit of Escherichia coli gamma-glutamyltranspeptidase

Sequence comparison showed that residues Thr407, Asp433, and Met464 in the small subunit of Escherichia coli gamma-glutamyltranspeptidase (EcGGT) were conserved in the aligned enzymes. In this study, we further investigated the functional significance of these conserved residues by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 cells and purified to near homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown no GGT activity under enzyme assay conditions and activity staining. Furthermore, mutations on these residues impaired the capability of autocatalytic processing of the enzyme. Based on these observations, it is concluded that these residues play an important role in the enzyme maturation.     

Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli

In Escherichia coli, thrA, metLM, and lysC encode aspartokinase isozymes that show feedback inhibition by threonine, methionine, and lysine, respectively. In vitro chemical mutagenesis of the cloned lysC gene was used to identify residues and regions of the polypeptide essential for feedback inhibition by lysine. The isolated lysine-insensitive mutants were demonstrated to have missense mutations in amino acid residues 323-352, and at position 250 of aspartokinase III.     

Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.     

Riboflavin synthase of Escherichia coli. Effect of single amino acid substitutions on reaction rate and ligand binding properties

Conserved amino acid residues of riboflavin synthase from Escherichia coli were modified by site-directed mutagenesis. Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins. S41A and H102Q mutants had substantially reduced reaction velocities. Replacements of various other conserved polar residues had little impact on catalytic activity. (19)F NMR protein perturbation experiments using a fluorinated intermediate analog suggest that the N-terminal sequence motif MFTG is part of one of the substrate-binding sites of the protein.     

Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Redox control of disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K+-independent (type II) H+-pyrophosphatases.     

Functions in outer and inner membranes of Escherichia coli for ferrichrome transport.

Mutants of the fhuA gene of Escherichia coli K-12, which encodes a receptor protein in the outer membrane, took up ferrichrome after exposure to pronase, whereas fhuB mutants remained transport negative. The latter finding supports our previous proposal that fhuB mutants are defective in a function that residues in the cytoplasmic membrane. Cells remained completely viable after treatment with pronase, although they became sensitive to the antibiotic actinomycin.     

Density gradient enrichment of Escherichia coli lpxL mutants

We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992-7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.     

Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.     

Antigenic determinants of murein lipoprotein and its exposure at the surface of Enterobacteriaceae.

Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Salmonella, Arizona, and Shigella antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti-lipoprotein titers than anti-R-lipopolysaccharide titers. Correspondingly,absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipolysaccharide on the cell surface. In wild-type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface.     

Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins

LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane. Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria. These Trp residues were mutagenized to obtain defective LolB mutants. Mutation of Trp at position 52 to Pro impaired the receptor activity and caused accumulation of the LolA-lipoprotein complex in the periplasm. Similar mutants were obtained for Trp at position 117. A mutant with Gly instead of Trp at position 148 retained the receptor activity but inhibited growth upon its overproduction. The outer membrane sorting of this mutant seemed to be defective, lipoprotein transfer thereby being perturbed when it was overproduced. Despite the strong conservation, no defective mutant for Trp at position 183 was obtained, and only weak mutants were isolated for Trp at position 18. Based on the crystal structure of LolB, the phenotypes of these mutants are discussed.     

Opening the KcsA K+ channel: tryptophan scanning and complementation analysis lead to mutants with altered gating

The properties of the KcsA channel were investigated using a combination of tryptophan scanning of the two transmembrane helices followed by random mutagenesis at targeted residues. The tryptophan mutants were subjected to two screens: oligomeric stability and ability to complement the K+ uptake deficiency of the TK2420 Escherichia coli strain. Oligomeric stability is affected primarily by mutations at sites that border on and interact with the selectivity filter, while the complementation assays identified residues at the crossing point of the inner helices. Sites identified by the complementation assay in the tryptophan screen were subjected to random mutagenesis and selection by complementation. We have found two mutants, A108S and A108T, which have dramatically increased open probability while retaining the basic property of oligomeric stability.     

Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis

The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.     

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.     

Delineation of the toxin coding fragments and an insect-specificity region of a dual toxicity Bacillus thuringiensis crystal protein gene

A series of deletion mutants have been constructed from the dual toxicity Bacillus thuringiensis aizawai IC1 (Bta IC1) crystal protein gene. The mutant toxin genes were expressed in Escherichia coli, their protein products purified and the authenticity of these mutant proteins confirmed immunologically. Analysis of the toxicity spectra of these mutants revealed that lepidopteran toxicity is located on the N-terminal region of the toxin between residues Ile30-Glu595. 3' deletion of a further 37 residues from Glu595 of the lepidopteran-specific toxin abolished lepidopteran toxicity but the resulting protein consisting of residues Ile30-Gly558 was still fully toxic to dipteran larvae and cells. Another mutant crystal protein gene truncated to encode residues between Ile30-Gly563 was toxic only to diptera. These data indicate that the determinants of lepidopteran specificity in the Bta IC1 toxin are located between residues Gly558-Glu595 and that the N-terminal portion of the toxin between Ile30-Gly558 is sufficient to express dipteran toxicity.     

Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.     

Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.