Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.     

Membrane-anchored cytochrome cy mediated microsecond time range electron transfer from the cytochrome bc1 complex to the reaction center in Rhodobacter capsulatus

In Rhodobacter capsulatus, the soluble cytochrome (cyt) c2 and membrane-associated cyt cy are the only electron carriers which operate between the photochemical reaction center (RC) and the cyt bc1 complex. In this work, cyt cy mediated microsecond time range electron transfer kinetics were studied by light-activated time-resolved absorption spectroscopy using a mutant strain lacking cyt c2. In intact cells and in isolated chromatophores of this mutant, only approximately 30% of the RCs had their photooxidized primary donor rapidly rereduced by cyt cy. Of these 30%, about half were reduced with a half-time of approximately 5 micros attributed to preformed complexes, and the other half with a half-time of approximately 40 micros attributed to cyt cy having to move from another site. This slower phase was affected by addition of glycerol, indicating its dependence on the viscosity of the medium. Cyt cy, despite its rereduction by ubihydroquinone oxidation in the millisecond time range, remained virtually unable to deliver electrons to other RCs which stayed photooxidized for several seconds. Furthermore, using two flashes separated by a variable time interval, it was shown that the fast electron donating complex was reformed in about 60 micros, a time span probably reflecting electron transfer from cyt c1 to cyt cy. In the absence of the cyt bc1 complex, the steady-state level of cyt cy in the chromatophore membranes obtained using cells grown in minimal medium was decreased to approximately 50%. The remaining cyt cy , however, was able to form the fast electron donating complex with the RC (half-time of approximately 5 micros), whereas the slower phase with a half-time of approximately 40 micros was strongly decelerated. This finding suggests a role for the cyt bc1 complex in stabilizing cyt cy and providing its "other" site, possibly via a close association between these components. Taken together, it is concluded that although cyt cy is present in substoichiometric amount compared to the RCs, it supports efficiently photosynthetic growth of R. capsulatus in the absence of cyt c2 because it can mediate fast electron transfer from the cyt bc1 complex to the RC during multiple turnovers of the cyclic electron flow.     

Characterization of the repeat-tract instability and mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader

The RFC1 gene encodes the large subunit of the yeast clamp loader (RFC) that is a component of eukaryotic DNA polymerase holoenzymes. We identified a mutant allele of RFC1 (rfc1::Tn3) from a large collection of Saccharomyces cerevisiae mutants that were inviable when present in a rad52 null mutation background. Analysis of rfc1::Tn3 strains indicated that they displayed both a mutator and repeat-tract instability phenotype. Strains bearing this allele were characterized in combination with mismatch repair (msh2Delta, pms1Delta), double-strand break repair (rad52), and DNA replication (pol3-01, pol30-52, rth1Delta/rad27Delta) mutations in both forward mutation and repeat-tract instability assays. This analysis indicated that the rfc1::Tn3 allele displays synthetic lethality with pol30, pol3, and rad27 mutations. Measurement of forward mutation frequencies in msh2Delta rfc1:Tn3 and pms1Delta rfc1:Tn3 strains indicated that the rfc1::Tn3 mutant displayed a mutation frequency that appeared nearly multiplicative with the mutation frequency exhibited by mismatch-repair mutants. In repeat-tract instability assays, however, the rfc1::Tn3 mutant displayed a tract instability phenotype that appeared epistatic to the phenotype displayed by mismatch-repair mutants. From these data we propose that defects in clamp loader function result in DNA replication errors, a subset of which are acted upon by the mismatch-repair system.     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb PstI fragment and a 4.8 kb PstI/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5' region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Photosynthetic deficiency of a pufX deletion mutant of Rhodobacter sphaeroides is suppressed by point mutations in the light-harvesting complex genes pufB or pufA

The pufX gene of the facultative phototroph Rhodobacter sphaeroides encodes a membrane protein that is required for photoheterotrophic growth. Deletion of pufX impairs the photosynthetic generation of a transmembrane potential, suggesting a role for the PufX protein in light-driven cyclic electron transfer [Farchaus, J. W., et al. (1992) EMBO J. 11, 2779-2788]. Here we describe the isolation and characterization of 65 spontaneous suppressor mutants in which photosynthetic competence was restored by secondary mutations. Genetic analysis revealed the occurrence of single point mutations altering highly conserved residues within the light-harvesting complex, B875. One of three tryptophan codons was changed to stop or arginine codons in 89% of these suppressor mutants. Spectral characterization and Western blot analysis were used to examine the B875 assembly and the stable expression of the altered light-harvesting polypeptides. Three different groups of suppressor mutants were found: (1) No stable expression of altered B875 polypeptides was detected for the alpha 43W-->* and beta 44W-->* mutants. (2) There was expression of the mutated B875-beta chain, but no stable B875 assembly in the beta 47W-->R mutants. (3) Intact B875 complexes were found for the alpha 47S-->F or beta 20H-->R mutants. These results provide evidence that the differently altered B875 polypeptides do not substitute directly for the PufX protein but lead to structural rearrangements in the macromolecular membrane organization, thus restoring a sufficiently high capacity for light-driven cyclic electron transfer.     

The Rhizobium etli rpoN locus: DNA sequence analysis and phenotypical characterization of rpoN, ptsN, and ptsA mutants

The rpoN region of Rhizobium etli was isolated by using the Bradyrhizobium japonicum rpoN1 gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258, rpoN, ORF191, and ptsN, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and ptsN are homologous to conserved ORFs found downstream from rpoN genes in other bacterial species. Unlike in most other microorganisms, rpoN and ORF191 are separated by approximately 1.6 kb. The R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of nifH, and the metabolism of C4-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the rpoN gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the ptsN gene resulted in a decrease of melanin synthesis and nifH expression. In a search for additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in ptsA, a gene homologous to the Escherichia coli gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA mutant also displayed reduced expression of nifH. The ptsN and ptsA mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C4-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that ptsN and ptsA are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.     

Effects of temperature and deltaGo on electron transfer from cytochrome c2 to the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides

The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center-cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force -deltaGo is varied over a range of 350 meV. The temperature and deltaGo dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy lambda = 0.96 +/- 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10(-4) eV to 2.5 10(-4) eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than approximately 200 cm(-1). An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon deltaGo, characterized by a similar value of lambda (0.8 +/- 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Isolation and properties of Escherichia coli mutants which are nonpermissive for the growth of phage lambda

By selecting survivors of lambda phage infection, mutants of Escherichia coli K12 that block reproduction cycle of the phage have been isolated. Fourteen of these phage-tolerant mutants (lam mutants) were chosen and characterized biochemically and genetically. It was shown that these mutants were tolerant to infection by all the lambdoid phages, except for few cases, but they were susceptible to infection by a non-lambdoid temperate phage (phi299), P1 or T phages. The mutants can be divided into at least three groups: (1) A mutant (lam 16) strain that seems to block normal penetration of phage DNA: (2) Three mutant (lam 64, lam 67 and lam 71) strains that block an "early" step(s) of phage growth, including phage DNA synthesis: (3) Six mutant (lam 24, lam 25, lam 26, lam 27, lam 646 and lam 6) strains that block normal functioning of the gene E products and produce unusual head structures. Some lambdoid phages and lambda mutants that overcome the interference by the lam mutations have been obtained, and were used as tools for characterizing the host mutations. Two (lam 12 and lam 13) mutant strains and one (lam 1) mutant were inferred as affecting the expression of "late" genes, and early gene, respectively, by this test.     

Mutational analysis of the genes encoding the photosystem II proteins in Cyanobacterium synechocystis sp. 6803

Chlorophyll--binding protein CP43 and cytochrome b559, encoded by psbC and psbE/F genes, are the components of photosystem II (PS II). Three psbC- and four psbE/F- mutants were isolated from the collection of PS II-deficient mutants of the cyanobacterium Synechocystis sp. 6803. Restoration of photosynthetic activity was achieved by transformation of psbE/F- mutants with cloned psbE/F gene cluster from wild type cells and each of psbC- mutants--with specific part of wild type psbC gene. DNA fragments carrying the mutations were isolated from mutant cells and sequenced. The mutations which affect PS II activity were identified in psbC gene as "frameshift" mutation, stop-codon formation, or as deletion of three nucleotides resulting in loss of one of three Phe residues in position 422-424 of CP43. Sequence of mutant psbE/F genes revealed single mutations resulting in deletion of Phe-36 or substitution of Pro-63 for Leu in alpha-subunit and Val-29 for Phe in beta-subunit of cytochrome b559.     

Mechanism of inhibition of electron transfer by amino acid replacement K362M in a proton channel of Rhodobacter sphaeroides cytochrome c oxidase

The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.     

In situ analysis of adhesion molecule expression in kidneys infected with murine malaria

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P(ow) 2.8) but they could grow in medium overlaid with organic solvents having a log P(ow) value higher than that of toluene such as p-xylene (log P(ow) 3.1), cyclohexane (log P(ow) 3.4) and n-hexane (log P(ow) 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3.7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.     

Value of image-guided needle aspiration cytology in the assessment of thoracolumbar and sacrococcygeal masses

We have used a primary cloning assay to determine the frequency of 6-thioguanine (TG)-resistant tubular epithelial cells in kidney tissue from 72 human donors ranging in age from 2 to 94 years. The frequency of TG-resistant mutants ranged from approximately 5 x 10(-5) for donors in the first decade of life to approximately 2.5 x 10(-4) for donors in the eighth and later decades of life. Two different statistical analyses indicated that this increase in mutant frequency is exponential with age. We also observed a 2-fold higher TG-resistant mutant frequency in nephrectomy kidneys containing a coincident renal carcinoma. DNA sequence analyses revealed HPRT gene mutations in each of 14 TG-resistant mutants from seven unrelated donors. Thirteen of these 14 mutants resulted from independent mutational events. These results suggest that somatic mutations are common in renal--and perhaps in other human--epithelia, and thus could play an important role in the genesis of age-associated disease.     

The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.     

Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier

We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide. Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding. In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL. The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs. This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine. In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta). This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle. Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type. The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.