Thesec andprl genes ofEscherichia coli

Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec,secretion). The other identifies dominant suppressors of signal sequence mutations (prl,proteinlocalization). Subsequent characterization reveals that in at least three cases,prlA/secY,prlD/secA, andprlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.     

Regulation of the Escherichia coli secA Gene Is Mediated by Two Distinct RNA Structural Conformations

Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X. SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation. Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution. The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus. The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence. The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus. The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence. As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene. The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain. Received: 30 July 1998 / Accepted: 18 September 1998     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> possesses two <Emphasis Type="Italic">secA</Emphasis> homologous genes that are essential for viability

SecA is a central component of the bacterial Sec preprotein translocase. Besides the housekeeping SecA (SecA1), some mostly pathogenic Gram-positive bacteria possess an accessory SecA (SecA2) that is involved in the export of a few substrates only. Here we show that neither of the two secA homologous genes present in the genome of the non-pathogenic bacterium Corynebacterium glutamicum can be deleted, unless a copy of the respective gene is provided in trans on a plasmid. This finding is in marked contrast to all other cases examined so far making C. glutamicum the first reported bacterium possessing two essential SecA proteins.     

Molecular typing of Coorg black pepper yellows phytoplasma by multiple gene analyses

In previous work, Coorg black pepper yellows phytoplasma (CBPYp), a ‘Candidatus Phytoplasma asteris'‐related strain, was identified in association with black pepper plants exhibiting yellows symptoms in southern India. In the present study, multiple gene (16S rRNA, tuf, rplV‐rpsC, secY and secA) sequence analyses were carried out for finer characterisation of CBPYp isolates identified in seven plants. Nucleotide sequences of each gene studied were identical among all the CBPYp isolates here analysed. Comparison of virtual restriction fragment length polymorphism (RFLP) patterns, validated by actual digestion of polymerase chain reaction (PCR) products, revealed that CBPYp is a member of subgroups 16SrI‐B, rpI‐L, tufI‐B, secYI‐L and secA1‐A. Interestingly, alignments of nucleotide sequences with other ‘Candidatus Phytoplasma asteris'‐related strains revealed the presence of CBPYp‐specific single nucleotide polymorphisms (SNPs), located in restriction sites for endonucleases not used for conventional classification. CBPYp‐specific SNPs in genes 16S rRNA, tuf and secA were detectable by virtual and actual RFLP assays, while SNPs present in rplV‐rpsC and secY genes were not located in any restriction recognition site. CBPYp‐specific SNPs can be used as molecular markers for the specific identification of CBPYp and for future research focused on investigating epidemiology and ecology of CBPYp in India.     

Molecular identification of ‘Candidatus Phytoplasma palmicola’ associated with coconut lethal yellowing in Equatorial Guinea

During the past two decades, a high mortality of coconut palms was observed in the coastal areas of Equatorial Guinea. Reportedly, the palm population has been reduced by 60%–70%, and coconut production has decreased accordingly. To identify the cause of the mortality, a survey was carried out in April 2021 in various localities of the coconut belt. Molecular analyses carried out on 16S rRNA and secA genes detected phytoplasma presence in the majority of the samples. Sequencing and BLAST search of the 16S rRNA gene sequences showed >99% identity of the detected phytoplasmas to ‘Candidatus Phytoplasma palmicola’. The RFLP analyses of 16S ribosomal gene using Tru1I and TaqI enzymes led to assign these phytoplasmas to subgroup 16SrXXII-A. In all samples that tested positive, including one from a hybrid coconut palm and two from oil palm the same phytoplasma was identified. The phylogenetic analyses of 16S rRNA and secA genes confirmed respectively 99.98%–100% and 97.94%–100% identity to ‘Ca. P. palmicola’. RFLP analyses using MboII enzyme on the secA gene amplicon differentiated the phytoplasma found in Equatorial Guinea from those present in Ghana and Ivory Coast. The Equatorial Guinean phytoplasma strain resulted to be identical to the strains from Mozambique, confirming the presence of a geographic differentiation among phytoplasma strains in the coastal areas of Western and Central Africa. The identified phytoplasma is different from the ‘Ca. P. palmicola’ strains found in Ghana and Ivory Coast and represents the first identification a 16SrXXII-A strain in Equatorial Guinea and in Central Africa. Strict monitoring and surveillance procedures for early detection of the pathogen are strongly recommended to reduce its impact and further spread in the country and permit the recovery of coconut plantations.     

Characterization of the Nuclear- and Plastid-Encoded secA-Homologous Genes in the Unicellular Red Alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.     

Regulation of Escherichia coli secA by Cellular Protein Secretion Proficiency Requires an Intact Gene X Signal Sequence and an Active Translocon

secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.     

Fission yeast sta mutations that stabilize an unstable minichromosome are novel cdc2-interacting suppressors and are involved in regulation of spindle dynamics

Cytological observations have shown that the presence of unstable minichromosomes can delay progression through the early stages of mitosis in fission yeast (Schizosaccharomyces pombe), suggesting that such minichromosomes may provide a useful tool for examining the system that regulates the coordinated segregation of chromosomes. One such unstable minichromosome is a large circular minichromosome. We previously showed that the mitotic instability of this minichromosome is probably due to the frequent occurrence of catenated forms of DNA after replication. To identify genes involved in the regulation of chromosome behavior in mitosis, we isolated mutants which stabilized this minichromosome. Three loci (stal, sta2, and sta3) were identified. Two of them were found to be suppressors of temperature-sensitive mutations in cdc2, which encodes the catalytic subunit of muturation promoting factor (MPF). They show no linkage to, and are thus different from, sucl, and cdc13, previously identified as genes that interact with cdc2. The other mutation mapped to a gene previously identified as being required for the correct formation of the mitotic spindle. Data provided in this study suggest that the sta genes are involved in the regulation of spindle dynamics to ensure proper chromosome segregation during mitosis.     

Cloning and expression of the secA gene of a marine bacterium,Vibrio alginolyticus,and analysis of its function in Escherichia coli

We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation. Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V. alginolyticus. It was shown to encode a 103.3-kDa protein. The deduced amino acid sequence of V. alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E. coli (EcSecA). The secA gene of E. coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V. alginolyticus secA. Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide. Expression of VaSecA in E. coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature. VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms. VaSecA was overproduced in V. alginolyticus and purified to homogeneity for N-terminal sequencing. The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA.     

Comparison of lantibiotic gene clusters and encoded proteins

Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions.The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding and ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system.Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisl and spal genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster.A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.     

A nonsense mutation in the structural gene for glutamine synthetase leading to loss of nitrogen regulation in Klebsiella aerogenes

Summary An amber mutation (glnA3711), the first nonsense mutation isolated in Klebsiella aerogenes, is described. When amber suppressors were present, the mutant made active glutamine synthetase which was more thermolabile than wild type, showing that glnA3711 lies in the structural gene for glutamine synthetase. Strains carrying the glnA3711 allele were unable to express nitrogen regulation of genes coding for histidase, asparaginase, and glutamate dehydrogenase unless amber suppressors were also present. These results support a model that expression of gene(s) from the glnA promoter is required for nitrogen regulation in K. aerogenes.     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary In the preceding paper of this series (Dujardin et al. 1980a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit ^- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific: it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cobbox gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit ^- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox 1) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit ^- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutants.Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit ^- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for nuclear accommodation of mitochondria) or in the mitochondrial genome (abbreviated MIM for mitochondrial-mitochondrial interaction).The specificity of action of various suppressors upon some 250 different mit ^- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit ^- mutations within the introns of this gene.Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene.Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.