Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.     

苏云金杆菌辅助蛋白基因p19和orf1-orf2对杀虫晶体蛋白Cyt1 Aa表达的影响(英文)

为检测苏云金杆菌辅助蛋白P19和ORF1 ORF2对杀虫晶体蛋白Cyt1Aa表达的影响 ,构建了 5个重组表达质粒。 5个质粒都含有cyt1Aa基因 ,但pT1只含有cyt1Aa基因 ,pT2同时含有p19基因 ,pT3同时含有orf1 orf2串联基因 ,pT4同时含有p19基因和p2 0基因 ,pT5同时含有orf1 orf2串联基因和p2 0基因。将这 5个表达质粒和质粒pWF4 5电转化到苏云金杆菌晶体缺陷型 4Q7中 ,分别获得转化菌株Bt T1、Bt T2、Bt T3、Bt T4、Bt T5和Bt WF4 5。SDS PAGE结果显示 ,菌株Bt T1、Bt T2和Bt T3只产生少量的 2 7kDCyt1Aa蛋白 ,而且部分降解为大约 2 4kD的蛋白。而Bt T4和Bt T5能产生大量的Cyt1Aa蛋白 ,但Bt T4和Bt T5的Cyt1Aa蛋白产量都明显少于Bt WF4 5。电镜观察和生物测定结果表明Bt T4和Bt T5与Bt WF4 5的晶体大小和杀蚊毒力没有显著性差异。研究表明P19和ORF1 ORF2对Cyt1Aa蛋白的合成显示可能有抑制作用。     

Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.     

Genes in the accessory sec locus of Streptococcus gordonii have three functionally distinct effects on the expression of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is strongly correlated with the expression of the large surface glycoprotein GspB. A 14 kb chromosomal region downstream of gspB was previously shown to be required for the expression of this protein. The region encodes SecA2 and SecY2, which are components of an accessory secretion system dedicated specifically to the export of GspB. The region also includes three genes (gly, nss and gtf) that encode proteins likely to function in carbohydrate metabolism, and four genes (orf1-4) that encode proteins of unknown function. In this report, we have investigated the role of these genes in GspB expression. We found that disruption of orf1, orf2 or orf3 resulted in a loss of GspB export and the intracellular accumulation of GspB. As they are apparently essential components of the accessory secretion system, these genes were renamed asp1-3 (for accessory secretory protein). In gtf and orf4 mutants, gspB was transcribed, but no GspB was detected. These results suggest that Gtf and Orf4 are required for the translation or for the stability of GspB. In contrast, gly and nss mutants were able to express and export GspB. However, disruption of these genes appeared to affect the carbohydrate composition of this glycoprotein. As asp1-3, gtf and orf4, but not gly and nss, are conserved in the accessory sec loci of several staphylococcal and streptococcal species, these genes may also have crucial roles in the expression and export of GspB homologues in the other Gram-positive bacteria.     

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.     

Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.     

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.     

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.     

A homologue of the 3-oxoacyl-(acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates virulence factors via N-acyl homoserine lactone and fatty acid synthesis

Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Deltaorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Deltaorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Deltaorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Deltaorf3 mutant. The phenotypes of the Deltaorf3 mutant and an AHL synthesis (DeltapsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Deltaorf3 mutant. The swarming motility of the Deltaorf3 mutant was greater than that of the DeltapsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Deltaorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605.