Generation of Campylobacter fetus S-layer protein diversity utilizes a single promoter on an invertible DNA segment

Wild-type strains of Campylobacter fetus contain a monomolecular array of surface layer proteins (SLPs) and vary the antigenicity of the predominant SLP expressed. Reciprocal recombination events among the eight genomic SLP gene cassettes, which encode 97- to 149 kDa SLPs, permit this variation. To explore whether SLP expression utilizes a single promoter, we created mutant bacterial strains using insertional mutagenesis by rescue of a marker from plasmids. Experimental analysis of the mutants created clearly indicates that SLP expression solely utilizes the single sapA promoter, and that for variation C. fetus uses a mechanism of DNA rearrangement involving inversion of a 6.2 kb segment of DNA containing this promoter. This DNA inversion positions the sapA promoter immediately upstream of one of two oppositely oriented SLP gene cassettes, leading to its expression. Additionally, a second mechanism of DNA rearrangement occurs to replace at least one of the two SLP gene cassettes bracketing the invertible element. As previously reported promoter inversions in prokaryotes, yeasts and viruses involve alternate expression of at most two structural genes, the ability of C. fetus to use this phenomenon to express one of multiple cassettes is novel.     

Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent.

Wild-type strains of Campylobacter fetus are covered by a monomolecular array of surface layer proteins (SLPs) critical for virulence. Each cell possesses eight SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97 to 149 kDa. Variation of SLP expression occurs by a mechanism of nested DNA rearrangement that involves the inversion of a 6.2-kb sapA promoter-containing element alone or together with one or more flanking SLP gene cassettes. The presence of extensive regions of identity flanking the 5' and 3' ends of each SLP gene cassette and of a Chi-like recognition sequence within the 5' region of identity suggests that rearrangement of SLP gene cassettes may occur by a generalized (RecA-dependent) homologous recombination pathway. To explore this possibility, we cloned C. fetus recA and created mutant strains by marker rescue, in which recA is disrupted in either S+ or S- strains. These mutants then were assessed for their abilities to alter SLP expression either in the presence or absence of a complementary shuttle plasmid harboring native recA. In contrast to all previously reported programmed DNA inversion systems, inversion in C. fetus is recA dependent.     

The invertible P-DNA segment in the chromosome of Escherichia coli.

In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region. This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences. The element e14 contains a gene which kills the host cell. This can be repressed by a function also coded by e14. The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element. The invertible segment and pin gene were sequenced. The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment. Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly). The complete DNA sequences of both invertible segments were screened for homology. No resemblance was found. The P segment is flanked by inverted repeat sequences of 16 bp. Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region. This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.     

DNA Inversions between Short Inverted Repeats in Escherichia Coli

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.     

Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

Molecular and catalytic properties of monoacetylphloroglucinol acetyltransferase from Pseudomonas sp. YGJ3

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl(-). Cl(-) and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M(r)=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG](2)) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.     

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.     

Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.     

Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis

Restriction endonucleases and agarose gel electrophoresis were used to analyze plasmid pLM2001, which is required for lactose metabolism by Streptococcus lactis LM0232. The enzymes XhoI, SstI, BamHI, and KpnI each cleaved the plasmid into two fragments, whereas EcoRI and BglII cleaved the plasmid into seven and five fragments, respectively. Sizing of fragments and multiple digestions allowed construction of a composite restriction map. The KpnI fragments of pLM2001 were cloned into the KpnI cleavage site of the vector plasmid pDB101. A recombinant plasmid (pSH3) obtained from a lactose-fermenting, erythromycin-resistant (Lac+ Eryr) transformant of Streptococcus sanguis Challis was analyzed by enzyme digestion and agarose gel electrophoresis. Plasmid pSH3 contained 7 of the 11 KpnI-HindIII fragments from pLM2001 and 5 of the 7 fragments from pDB101. It was determined that a 23-kilobase (kb) KpnI-generated fragment from pLM2001 had been cloned into pDB101 with deletion of part of the vector plasmid. The recombinant plasmid could be transformed with high frequency into several Lac- strains of S. sanguis, conferring the ability to ferment lactose and erythromycin resistance. The presence of pSH3 allowed a strain deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-dependent phosphotransferase system to efficiently ferment lactose. Under conditions designed to maximize curing of plasmid DNA with acriflavin, no Lac- derivatives could be isolated from cells transformed with pSH3. Seven of the 40 Lac+ colonies isolated after 10 transfers in acriflavin were shown to be sensitive to erythromycin and did not appear to harbor plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the lactose-metabolizing genes from Streptococcus lactis.

Restriction endonucleases and agarose gel electrophoresis were used to analyze plasmid pLM2001, which is required for lactose metabolism by Streptococcus lactis LM0232. The enzymes XhoI, SstI, BamHI, and KpnI each cleaved the plasmid into two fragments, whereas EcoRI and BglII cleaved the plasmid into seven and five fragments, respectively. Sizing of fragments and multiple digestions allowed construction of a composite restriction map. The KpnI fragments of pLM2001 were cloned into the KpnI cleavage site of the vector plasmid pDB101. A recombinant plasmid (pSH3) obtained from a lactose-fermenting, erythromycin-resistant (Lac+ Eryr) transformant of Streptococcus sanguis Challis was analyzed by enzyme digestion and agarose gel electrophoresis. Plasmid pSH3 contained 7 of the 11 KpnI-HindIII fragments from pLM2001 and 5 of the 7 fragments from pDB101. It was determined that a 23-kilobase (kb) KpnI-generated fragment from pLM2001 had been cloned into pDB101 with deletion of part of the vector plasmid. The recombinant plasmid could be transformed with high frequency into several Lac- strains of S. sanguis, conferring the ability to ferment lactose and erythromycin resistance. The presence of pSH3 allowed a strain deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-dependent phosphotransferase system to efficiently ferment lactose. Under conditions designed to maximize curing of plasmid DNA with acriflavin, no Lac- derivatives could be isolated from cells transformed with pSH3. Seven of the 40 Lac+ colonies isolated after 10 transfers in acriflavin were shown to be sensitive to erythromycin and did not appear to harbor plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene''s expression.

Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process.     

Chromatin structure at the 3'-boundary of the human beta-globin locus control region hypersensitive site-2

Chromatin structure was examined at the 3′-boundary region of the human β-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif—this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3′-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site.