A Protein (M9) Associated with Monoclonal Antibody-Mediated Agglutination of Mycoplasma gallisepticum Is a Member of the pMGA Family

ABSTRACT A 62-kDa cell surface antigen (M9) of Mycoplasma gallisepticum PG31 that mediates antibody-induced agglutination of the organism was purified and subjected to N-terminal amino-acid sequencing. A 999-bp region of the cDNA encoding the M9 protein was generated by reverse transcription-PCR, and its nucleotide sequence was determined. PCR primers based on this sequence were used to screen a genomic DNA library of PG31. A full-length M9 protein-encoding gene was isolated and sequenced, revealing 96% nucleotide identity with thepMGA1.1 gene of M. gallisepticum S6. Sequence analyses of the M9 gene and flanking open reading frames that encode other pMGA family members suggest that a tandemly repeated GAA sequence may influencepMGA gene expression.

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Ferrous Iron- and Sulfur-Induced Genes in Sulfolobus metallicus

Applied and Environmental Microbiology ◽

10.1128/aem.02589-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2491-2497 ◽

Cited By ~ 63

Author(s):

Stephan Bathe ◽

Paul R. Norris

Keyword(s):

Reverse Transcription ◽

Ferrous Iron ◽

Subtractive Hybridization ◽

Open Reading Frames ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Genes Encoding ◽

Induced Genes ◽

Reading Frames ◽

Sulfur Oxygenase Reductase

ABSTRACT Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

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Application of Recombinase-Based In Vivo Expression Technology to Bifidobacterium longum subsp. longum for Identification of Genes Induced in the Gastrointestinal Tract of Mice

Microorganisms ◽

10.3390/microorganisms8030410 ◽

2020 ◽

Vol 8 (3) ◽

pp. 410 ◽

Cited By ~ 2

Author(s):

Hiroka Koguchi ◽

Natsumi Ishigami ◽

Mikiyasu Sakanaka ◽

Kako Yoshida ◽

Sayaka Hiratou ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Gut Microbiota ◽

Bifidobacterium Longum ◽

Bacteriophage P1 ◽

Dna Library ◽

Reverse Transcription Pcr ◽

Specific Induction ◽

In Vivo Expression ◽

Genomic Dna Library

Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.

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Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

Journal of Bacteriology ◽

10.1128/jb.186.14.4730-4739.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4730-4739 ◽

Cited By ~ 60

Author(s):

Andrea K. White ◽

William W. Metcalf

Keyword(s):

Insertion Site ◽

Pseudomonas Stutzeri ◽

Gene Organization ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

E Coli ◽

Reverse Transcription Pcr ◽

Transposon Insertion ◽

Intergenic Sequences ◽

Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

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Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation

Applied and Environmental Microbiology ◽

10.1128/aem.00616-07 ◽

2007 ◽

Vol 73 (14) ◽

pp. 4477-4483 ◽

Cited By ~ 50

Author(s):

Ying-Fei Ma ◽

Jian-Feng Wu ◽

Sheng-Yue Wang ◽

Cheng-Ying Jiang ◽

Yun Zhang ◽

...

Keyword(s):

Nucleotide Sequence ◽

Resistance Genes ◽

Gene Deletion ◽

Degradation Pathway ◽

Open Reading Frames ◽

Mercury Resistance ◽

Dna Molecules ◽

Chromate Resistance ◽

Reverse Transcription Pcr ◽

Reading Frames

ABSTRACT The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1β group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1β plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.

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Membrane-Bound, 2-Keto-d-Gluconate-Yielding d-Gluconate Dehydrogenase from “Gluconobacter dioxyacetonicus” IFO 3271: Molecular Properties and Gene Disruption

Applied and Environmental Microbiology ◽

10.1128/aem.00493-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6551-6556 ◽

Cited By ~ 25

Author(s):

Hirohide Toyama ◽

Naoko Furuya ◽

Ittipon Saichana ◽

Yoshitaka Ano ◽

Osao Adachi ◽

...

Keyword(s):

Double Mutant ◽

Culture Medium ◽

Wild Type Strain ◽

Reductase Activity ◽

Pcr Primers ◽

Open Reading Frames ◽

Pcr Product ◽

Mutant Strains ◽

Membrane Bound ◽

Reading Frames

ABSTRACT Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from “Gluconobacter dioxyacetonicus” IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-δ-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that “G. dioxyacetonicus” IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.

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Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.733-741.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 733-741 ◽

Cited By ~ 29

Author(s):

Cécile J. B van der Vlugt-Bergmans ◽

Mariët J. van der Werf

Keyword(s):

Ring Opening ◽

Biochemical Characterization ◽

Rhodococcus Erythropolis ◽

Open Reading Frames ◽

Apparent Homogeneity ◽

Terminal Amino ◽

Acyl Coenzyme A ◽

Reading Frames

ABSTRACT A monoterpene ɛ-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB(monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.

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Transcriptional Organization of the czcHeavy-Metal Homeostasis Determinant from Alcaligenes eutrophus

Journal of Bacteriology ◽

10.1128/jb.181.8.2385-2393.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2385-2393 ◽

Cited By ~ 67

Author(s):

Cornelia Große ◽

Gregor Grass ◽

Andreas Anton ◽

Sylvia Franke ◽

Alexander Navarrete Santos ◽

...

Keyword(s):

Response Regulator ◽

Regulatory System ◽

Alcaligenes Eutrophus ◽

Northern Blotting ◽

Open Reading Frames ◽

Mrna Synthesis ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Reporter Strains ◽

Reading Frames

ABSTRACT The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5′ end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotideczcI message were found, and the presence of 6,200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493–503, 1997) was confirmed. Induction ofczcN, czcI, czcCBA, andczcDRS followed a similar pattern: transcription was induced best by 300 μM zinc, less by 300 μM cobalt, and only slightly by 300 μM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN toczcC and from czcD to czcS, but not between czcA and czcD nor betweenczcS and a 131-amino-acid open reading frame followingczcS. The CzcR putative response regulator was purified and shown to bind in the 5′ region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were ΔczcR and ΔczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the β-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

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Open reading frames flanking the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli

Gene ◽

10.1016/0378-1119(94)90647-5 ◽

1994 ◽

Vol 151 (1-2) ◽

pp. 153-156 ◽

Cited By ~ 2

Author(s):

JoséM. Galindo ◽

Gabriel Guarneros ◽

Francisco M. De La Vega

Keyword(s):

Escherichia Coli ◽

Open Reading Frames ◽

Encoding Gene ◽

Reading Frames

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An Alternative Succinate (2-Oxoglutarate) Transport System in Rhizobium tropici Is Induced in Nodules of Phaseolus vulgaris

Journal of Bacteriology ◽

10.1128/jb.00252-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5057-5067 ◽

Cited By ~ 10

Author(s):

Silvia Batista ◽

Eduardo J. Patriarca ◽

Rosarita Tatè ◽

Gloria Martínez-Drets ◽

Paul R. Gill

Keyword(s):

Phaseolus Vulgaris ◽

Carbon Source ◽

Sequence Similarity ◽

Optimal Growth ◽

Open Reading Frames ◽

Uptake System ◽

Rhizobium Tropici ◽

High Sequence Similarity ◽

Encoding Gene ◽

Reading Frames

ABSTRACT The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C4-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

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Functional Genomic Analysis of Two Staphylococcus aureus Phages Isolated from the Dairy Environment

Applied and Environmental Microbiology ◽

10.1128/aem.01864-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7663-7673 ◽

Cited By ~ 35

Author(s):

Pilar García ◽

Beatriz Martínez ◽

José María Obeso ◽

Rob Lavigne ◽

Rudi Lurz ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Dna Recombination ◽

Point Mutations ◽

Genomic Analysis ◽

Open Reading Frames ◽

Functional Genomic Analysis ◽

Replication Point ◽

Terminal Amino ◽

Functional Analyses ◽

Reading Frames

ABSTRACT The genomes of the two lytic mutant Staphylococcus aureus bacteriophages, vB_SauS-phiIPLA35 (phiIPLA35) and vB_SauS-phiIPLA88 (phiIPLA88), isolated from milk have been analyzed. Their genomes are 45,344 bp and 42,526 bp long, respectively, and contain 62 and 61 open reading frames (ORFS). Enzymatic analyses and sequencing revealed that the phiIPLA35 DNA molecule has 3′-protruding cohesive ends (cos) 10 bp long, whereas phiIPLA88 DNA is 4.5% terminally redundant and most likely is packaged by a headful mechanism. N-terminal amino acid sequencing, mass spectrometry, bioinformatic analyses, and functional analyses enabled the assignment of putative functions to 58 gene products, including DNA packaging proteins, morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, modification, and replication. Point mutations in their lysogeny control-associated genes explain their strictly lytic behavior. Muralytic activity associated with other structural components has been detected in virions of both phages. Comparative analysis of phiIPLA35 and phiIPLA88 genome structures shows that they resemble those of φ12 and φ11, respectively, both representatives of large genomic groupings within the S. aureus-infecting phages.

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