Effects of photosynthetic reaction center H protein domain mutations on photosynthetic properties and reaction center assembly in Rhodobacter sphaeroides

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane alpha-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex.     

Comparison of reaction centers from Rhodobacter sphaeroides and Rhodopseudomonas viridis: overall architecture and protein-pigment interactions

Photosynthetic reaction centers (RCs) from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis are protein complexes closely related in both structure and function. The structure of the Rps. viridis RC was used to determine the structure of the RC from Rb. sphaeroides. Small but meaningful differences between the positions of the helices and the cofactors in the two complexes were identified. The distances between helices AL and AM, between BL and BM, and between bacteriopheophytins BPL and BPM are significantly shorter in Rps. viridis than they are in Rb. sphaeroides RCs. There are a number of differences in the amino acid residues that surround the cofactors; some of these residues form hydrogen bonds with the cofactors. Differences in chemical properties and location of these residues account in some manner for the different spectral properties of the two RCs. In several instances, the hydrogen bonds, as well as the apparent distances between the histidine ligands and the Mg atoms of the bacteriochlorophylls, were found to significantly differ from the Rb. sphaeroides RC structure previously described by Yeates et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7993-7997] and Allen et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8487-8491].     

Different mechanisms of the binding of soluble electron donors to the photosynthetic reaction center of Rubrivivax gelatinosus and Blastochloris viridis

The tetraheme cytochrome subunits of the photosynthetic reaction centers (RCs) in two species of purple bacteria, Rubrivivax gelatinosus and Blastochloris (Rhodopseudomonas) viridis, were compared in terms of their capabilities to bind different electron-donor proteins. The wild-type RCs from both species and mutated forms of R. gelatinosus RCs (with amino acid substitutions introduced to the binding domain for electron-donor proteins) were tested for their reactivity with soluble cytochromes and high potential iron-sulfur protein. Cytochromes from both species were good electron donors to the B. viridis RC and the R. gelatinosus RC. The reactivity in the R. gelatinosus RC showed a clear dependence on the polarity of the charges introduced to the binding domain, indicating the importance of the electrostatic interactions. In contrast, high potential iron-sulfur protein, presumed to operate according to the hydrophobic mechanism of binding, reacted significantly only with the R. gelatinosus RC. Evolutionary substitution of amino acids in a region of the binding domain on the cytochrome subunit surface probably caused the change in the principal mode of protein-protein interactions in the electron-transfer chains.     

Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides.

Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed. It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26). The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale. However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure. The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins. A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale. Amino acids have been concluded to have no significant preference to localisation in transmembrane segments. Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.     

The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.     

Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences

In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.     

Comparative analysis of the primary structure of the reaction center-bound cytochrome subunit in purple bacteria

The amino acid sequences of the reaction center-bound cytochrome subunit of six species of purple bacteria were compared. Amino acid residues thought to be important in controlling the redox midpoint potentials of four hemes in Blastochloris (Rhodopseudomonas) viridis were found to be well conserved. As opposed to all other species studied, the amino acid sequence of the cytochrome subunit of B. viridis had several insertions of more than 10 residues at specific regions close to the LM core, suggesting that interaction of the cytochrome subunit with the LM core in most species is different from that in B. viridis. Distribution of charged amino acid residues on the surface of the cytochrome subunit was compared among six species and discussed from the viewpoint of interaction with soluble electron donors.     

Can contact potentials reliably predict stability of proteins?

The simplest approximation of interaction potential between amino acid residues in proteins is the contact potential, which defines the effective free energy of a protein conformation by a set of amino acid contacts formed in this conformation. Finding a contact potential capable of predicting free energies of protein states across a variety of protein families will aid protein folding and engineering in silico on a computationally tractable time-scale. We test the ability of contact potentials to accurately and transferably (across various protein families) predict stability changes of proteins upon mutations. We develop a new methodology to determine the contact potentials in proteins from experimental measurements of changes in protein's thermodynamic stabilities (DeltaDeltaG) upon mutations. We apply our methodology to derive sets of contact interaction parameters for a hierarchy of interaction models including solvation and multi-body contact parameters. We test how well our models reproduce experimental measurements by statistical tests. We evaluate the maximum accuracy of predictions obtained by using contact potentials and the correlation between parameters derived from different data-sets of experimental (DeltaDeltaG) values. We argue that it is impossible to reach experimental accuracy and derive fully transferable contact parameters using the contact models of potentials. However, contact parameters may yield reliable predictions of DeltaDeltaG for datasets of mutations confined to the same amino acid positions in the sequence of a single protein.     

Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

The 'light' and 'medium' subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the genes, nucleotide and amino acid sequence

The 'light' (L) and the 'medium' (M) subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis were isolated and their amino-terminal sequences, as well as the sequences of several chymotryptic peptides, determined. Rps. viridis DNA was cloned in the Escherichia coli plasmid pBR322. Mixed oligonucleotide probes derived from the amino acid sequences were synthesized and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the alpha and beta subunits of the light-harvesting complex and part of the gene for the reaction centre cytochrome. The nucleotide sequences of the L and M subunit genes and teh derived amino acid sequences are presented. The L subunit consists of 273 amino acids and has a mol. wt of 30 571. The M subunit consists of 323 amino acids and has a mol. wt of 35 902. The primary structure is discussed in the light of the recently published secondary and tertiary structure which has shown that both subunits contain five membrane-spanning helices.     

Diversity among B6 strains of Agrobacterium tumefaciens.

A total of 20 laboratory substrains of Agrobacterium tumefaciens strain B6 were compared with respect to six characteristics, including 3-ketolactose production, lysogeny, octopine catabolism, tumorigenic host range, and plasmid content. Within this group of strains diversity was found for all characteristics except 3-ketolactose production. Six substrains were lysogenized with an omega-type phage, whereas one substrain appeared neither sensitive to nor lysogenized with this bacteriophage. All but two substrains catabolized octopine and induced tumors on carrot disks. These 18 substrains harbor deoxyribonucleic acid sequences homologous to pTiB6-806. The two substrains unable to catabolize octopine were nontumorigenic and lacked detectable Ti plasmid sequences. Of the 20 substrains, 13 also contained sequences homologous to the cryptic plasmid pAtB6-806; 2 of the 18 substrains tumorigenic on carrots failed to induce tumors on Kalanchoe leaves. Their inability to induced tumors on this host, could not be correlated with lysogeny, with the presence or absence of pAtB6-806, or with the very large cryptic plasmid recently described. The Ti plasmids from these two strains were indistinguishable from pTiB6-806 by restriction enzyme analysis and could genetically convert a cured A. tumefaciens strain to tumorigenicity on both plant species. The results with these two strains suggest that parameters of tumorigenicity, such as host range, may be controlled by the bacterial chromosome.     

The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT–con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT–con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGP Pc 2) and the other from suspension cultures of Nicotiana alata (AGP Na 2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGP Pc 2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGP Na 2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.     

Functional analysis of an α-helical antimicrobial peptide derived from a novel mouse defensin-like gene

Gene-encoded antimicrobial peptides (AMPs) are an essential component of the innate immune system in many species. Analysis of β-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the β-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse β-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection.     

Rabbit phosphoglucose isomerase/neuroleukin/autocrine motility factor: cloning via interspecies identity

Phosphoglucose isomerase is the first committed enzyme of glycolysis. The protein also has a variety of biological activities on mammalian cells. The molecular basis of these extracellular functions is unclear, and the high resolution three-dimensional structure of a mammalian enzyme has not been described. We report here the cDNA and protein sequence for phosphoglucose isomerase from rabbit muscle. The sequence was obtained directly by PCR without the need to screen clones from a cDNA library and encoded active enzyme when expressed in bacterial cells. The 558 amino acid rabbit coding sequence is the same length as and highly similar (92% residue identity) to the sequences from human and pig and less so (88%) to the mouse enzyme. Non-conservative amino acid changes between the four mammalian sequences are concentrated in the first 35 and last five residues. The rabbit protein has an additional Cys residue and amino acid changes at five positions otherwise invariant in the mammalian enzymes.