scholarly journals All Seven comG Open Reading Frames Are Required for DNA Binding during Transformation of CompetentBacillus subtilis

1998 ◽  
Vol 180 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Y. S. Chung ◽  
D. Dubnau
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Cell Surface ◽  
Open Reading Frames ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Gram Negative ◽  
Transposon Insertions ◽  
Reading Frames

ABSTRACT The seven proteins encoded by the comG operon ofBacillus subtilis exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in comG result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed.

scholarly journals Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria

2001 ◽  
Vol 67 (4) ◽  
pp. 1959-1963 ◽  
Author(s):  
Claire A. Woodall ◽  
Karen L. Warner ◽  
Ronald S. Oremland ◽  
J. Colin Murrell ◽  
Ian R. McDonald
Keyword(s):  
Energy Source ◽  
Methyl Bromide ◽  
Open Reading Frames ◽  
Binding Motif ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
High Sequence Homology ◽  
Methyl Halide ◽  
High Degree ◽  
Reading Frames

ABSTRACT Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmugene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partialmetF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif. However,cmuB, identified in M. chloromethanicumand H. chloromethanicum, was not detected in IMB-1.

scholarly journals Two-Partner Secretion Systems of Neisseria meningitidis Associated with Invasive Clonal Complexes

2008 ◽  
Vol 76 (10) ◽  
pp. 4649-4658 ◽  
Author(s):  
Peter van Ulsen ◽  
Lucy Rutten ◽  
Moniek Feller ◽  
Jan Tommassen ◽  
Arie van der Ende
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Open Reading Frames ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Gram Negative ◽  
System 1 ◽  
Related Proteins ◽  
Encoding Genes ◽  
Reading Frames

ABSTRACT The two-partner secretion (TPS) pathway is widespread among gram-negative bacteria and facilitates the secretion of very large and often virulence-related proteins. TPS systems consist of a secreted TpsA protein and a TpsB protein involved in TpsA transport across the outer membrane. Sequenced Neisseria meningitidis genomes contain up to five TpsA- and two TpsB-encoding genes. Here, we investigated the distribution of TPS-related open reading frames in a collection of disease isolates. Three distinct TPS systems were identified among meningococci. System 1 was ubiquitous, while systems 2 and 3 were significantly more prevalent among isolates of hyperinvasive clonal complexes than among isolates of poorly invasive clonal complexes. In laboratory cultures, systems 1 and 2 were expressed. However, several sera from patients recovering from disseminated meningococcal disease recognized the TpsAs of systems 2 and 3, indicating the expression of these systems during infection. Furthermore, we showed that the major secreted TpsAs of systems 1 and 2 depend on their cognate TpsBs for transport across the outer membrane and that the system 1 TpsAs undergo processing. Together, our data indicate that TPS systems may contribute to the virulence of N. meningitidis.

scholarly journals High-Affinity Interaction between Gram-Negative Flagellin and a Cell Surface Polypeptide Results in Human Monocyte Activation

2000 ◽  
Vol 68 (10) ◽  
pp. 5525-5529 ◽  
Author(s):  
Patrick F. McDermott ◽  
Federica Ciacci-Woolwine ◽  
James A. Snipes ◽  
Steven B. Mizel
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Hypervariable Region ◽  
Gram Negative Bacteria ◽  
Human Monocytes ◽  
Necrosis Factor Alpha ◽  
Potent Inducer ◽  
Gram Negative ◽  
High Affinity ◽  
Tnf Α

ABSTRACT Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176–5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis,S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-α production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-α production (EC50) was obtained with 1.5 × 10−11 M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-α-inducing activity of the protein. Although less active than the wild-type protein, a Salmonellaflagellin mutant composed of only the central hypervariable region retained substantial TNF-α-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-α-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

scholarly journals Lysis Genes of the Bacillus subtilisDefective Prophage PBSX

1998 ◽  
Vol 180 (8) ◽  
pp. 2110-2117 ◽  
Author(s):  
Susanne Krogh ◽  
Steen T. Jørgensen ◽  
Kevin M. Devine
Keyword(s):  
Functional Analysis ◽  
Host Cell ◽  
Expression System ◽  
Cell Lysis ◽  
Lethal Gene ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Exported Protein

ABSTRACT Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they arexepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show thatxepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression ofxhlB (encoding the putative holin) together withxlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

scholarly journals A Novel DNA Polymerase Family Found inArchaea

1998 ◽  
Vol 180 (8) ◽  
pp. 2232-2236 ◽  
Author(s):  
Yoshizumi Ishino ◽  
Kayoko Komori ◽  
Isaac K. O. Cann ◽  
Yosuke Koga
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Open Reading Frames ◽  
Gene Products ◽  
Polymerase Gene ◽  
Hyperthermophilic Archaeon ◽  
Dna Polymerase Ii ◽  
Reading Frames

ABSTRACT One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes in M. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

scholarly journals Molecular Organization of Intrinsic Restriction and Modification Genes BsuM of Bacillus subtilis Marburg

2002 ◽  
Vol 184 (2) ◽  
pp. 381-389 ◽  
Author(s):  
Hideyuki Ohshima ◽  
Satoshi Matsuoka ◽  
Kei Asai ◽  
Yoshito Sadaie
Keyword(s):  
Bacillus Subtilis ◽  
Dna Methyltransferases ◽  
Open Reading Frames ◽  
Molecular Organization ◽  
Transcriptional Analysis ◽  
Logarithmic Phase ◽  
Modification System ◽  
Target Sequences ◽  
Reading Frames ◽  
Classical Mutant

ABSTRACT Transcriptional analysis and disruption of five open reading frames (ORFs), ydiO, ydiP, ydiR, ydiS, and ydjA, in the prophage 3 region of the chromosome of Bacillus subtilis Marburg revealed that they are component genes of the intrinsic BsuM restriction and modification system of this organism. The classical mutant strain RM125, which lacks the restriction and modification system of B. subtilis Marburg, lacks the prophage 3 region carrying these five ORFs. These ORFs constitute two operons, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon, both of which are expressed during the logarithmic phase of growth. The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases. The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known. Disruption of the ydiR-ydiS-ydjA operon resulted in enhanced transformation by plasmid DNA carrying multiple BsuM target sequences. Disruption of ydiO or ydiP function requires disruption of at least one of the following genes on the chromosome: ydiR, ydiS, and ydjA. The degrees of methylation of the BsuM target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with XhoI (an isoschizomer of BsuM) of DNAs extracted from the disruptant strains. Six XhoI (BsuM) sites were examined. XhoI digested at the XhoI sites in the DNAs from disruptants with disruptions in both operons, while XhoI did not digest at the XhoI sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the ydiR-ydiS-ydjA operon. Therefore, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon are considered operons that are responsible for BsuM modification and BsuM restriction, respectively.

scholarly journals Crystal Structure of F-93 from Sulfolobus Spindle-Shaped Virus 1, a Winged-Helix DNA Binding Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11544-11550 ◽  
Author(s):  
Paul Kraft ◽  
Andrea Oeckinghaus ◽  
Daniel Kümmel ◽  
George H. Gauss ◽  
John Gilmore ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Hot Springs ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Target Sequence ◽  
Winged Helix ◽  
Reading Frame ◽  
Biochemical Analyses ◽  
Reading Frames

ABSTRACT Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH ≤4.0; temperature of ≥70°C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-Å crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

scholarly journals An O-Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus

2008 ◽  
Vol 52 (10) ◽  
pp. 3580-3588 ◽  
Author(s):  
Vidya Dhote ◽  
Shuchi Gupta ◽  
Kevin A. Reynolds
Keyword(s):  
Ribosomal Subunit ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Naturally Occurring ◽  
Insertional Inactivation ◽  
Pathway Regulation ◽  
Reading Frames

ABSTRACT The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (Km = 30 ± 4 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP (Km = 200 ± 20 μM) or GTP (Km = 350 ± 60 μM) with a k cat of 2.2 ± 0.1 min−1. The phosphorylated HA is inactive against HA-sensitive ΔtolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k cat and Km values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5‴-dihydrohygromycin A and 5‴-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.

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