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PubMed1998

Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci.

A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5' and 3' ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

Base Sequence↗

PubMed2001

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Bacteria↗

PubMed2002

Identification, characterization, and expression of a second, bicistronic, operon involved in the production of lactocin S in Lactobacillus sakei L45.

Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed lasXY, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (lasA-W) and is transcribed in the opposite direction. The proximal of the two newly identified genes, lasX, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the lasX mutants have a strongly reduced level of lasA and lasA-W mRNA, thus indicating the likely cause of the Bac(-) phenotype of these mutants. The second ORF in the operon, lasY, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the lasXY operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the -35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of lasA and downstream genes.

Amino Acid Sequence↗

PubMed2003

Real-time reverse transcription-PCR analysis of expression of halobenzoate and salicylate catabolism-associated operons in two strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa JB2 can use 2-chlorobenzoate (2-CBa), 3-CBa, 2,3-dichlorobenzoate (2,3-DCBa), and 2,5-DCBa as sole carbon and energy sources, whereas strain 142 can only grow on 2-CBa and 2,4-DCBa. Both strains, however, harbor the same halobenzoate 1,2-dioxygenase (ohbAB) and chlorocatechol (clcABD) degradation genes necessary for the metabolism of ortho-CBas. In addition, the hybABCD operon, encoding a salicylate 5-hydroxylase, is also found in both strains. The expression of ohbAB, hybABCD, and clcABD operons was measured in cultures grown on different CBas as the sole carbon source and also in glucose-grown cells supplemented with CBas as inducers. A method to standardize real-time reverse transcription-PCR experimental data was used that allows the comparison of semiquantitative mRNA accumulation in different strains and culture conditions. In both strains, the ohb and hyb systems were induced in cells grown on 2-CBa or DCBas, whereas clc was induced only by DCBas. Repression by catabolite was observed both on ohb and clc systems in glucose-grown cells. Chlorocatechol 1,2-dioxygenase activity in JB2 was detected even in clc-repressed conditions, confirming the presence of additional isofunctional genes previously detected in P. aeruginosa 142. Although similar levels of induction of ohbAB were observed in strain JB2 grown on either benzoate, monochlorobenzoates, or DCBas, the ohbAB operon of strain 142 was only strongly induced by growth on 2-CBa and, to a lesser extent, on 2,4-DCBa. This observation suggests that regulation of the ohbAB operon may be different in both strains. The concomitant induction of ohb and hyb by CBas may allow the formation of hybrid halobenzoate dioxygenase(s) composed of Ohb/Hyb dioxygenase subunits and Hyb ferredoxin/ferredoxin reductase components.

Bacterial Proteins↗

PubMed2004

Characterization of the arginine deiminase operon of Streptococcus rattus FA-1.

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a sigma(70)-like promoter was mapped 5' to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.

Base Sequence↗

PubMed2004

Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny.

The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.

Bacterial Proteins↗

PubMed1989

Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherichia coli.

The structurally related pap and prs operons of the uropathogenic Escherichia coli isolate J96 encode a P and an F adhesin that mediate bacterial attachment to the human P blood group antigen and the Forssman antigen, respectively. Using probes prepared from different segments of the pap operon, Southern blot hybridizations were performed to characterize pap-related sequences of 30 E. coli clinical isolates expressing different adhesin phenotypes. Gene clusters encoding P and F adhesins displayed no restriction site polymorphism in sequences homologous to the papH, papC, and papD genes that encode proteins essential to the transport and polymerization of the subunits of the P-pilus adhesin. In contrast, pap-related genetic elements associated with a null phenotype either lacked homology to the papH, papC, and papD genes or displayed a restriction site polymorphism in this region. Sequences within and surrounding the J96 papG and prsG adhesin genes that determine the binding specificities to the P and F antigens, respectively, were not conserved. However, gene clusters encoding different binding specificities could not be distinguished based on such restriction site polymorphisms. The majority of clinical isolates had more than one copy of pap-related sequences that involved gene clusters similar to the J96 pap operon, as well as genetic elements that were related only to a part of this operon. The implications of this unexpected copy number polymorphism with respect to possible recombination events involving pap-related sequences are discussed.

Adhesins, Escherichia coli↗

PubMed1989

Mutations in the hemolytic-phospholipase C operon result in decreased virulence of Pseudomonas aeruginosa PAO1 grown under phosphate-limiting conditions.

The phospholipase C (PLC) operon of Pseudomonas aeruginosa consists of plcS, which encodes a heat-labile secreted hemolysin, and two in-phase, overlapping genes, plcR1 and plcR2, which may encode Pi-regulatory genes. A 2.8-kilobase-pair deletion mutation in this operon was constructed, and a tetracycline resistance (Tcr) cartridge replaced the deleted sequences. A deletion mutant of strain PAO1 was obtained through recombination between the flanking regions of the mutated cloned PLC operon and the homologous chromosomal regions. The deletion of the chromosomal PLC operon and its replacement by the Tcr cartridge was confirmed by Southern hybridization. The deletion strain, PLC SR, is nonhemolytic. However, it retains PLC activity when measured on a synthetic substrate. A second mutant strain, PLC R, contains a deletion in the plcR genes. This mutant is more hemolytic and produces more enzymatic activity than PAO1. The virulence of both of these mutants was compared with that of PAO1 in the mouse burn model of infection. When mice were infected with cultures grown in a high-Pi medium, there was a 10-fold increase in the 50% lethal dose of the mutants compared with PAO1. In contrast, when the inoculum originated from low-Pi cultures, there was a 200- to 10,000-fold increase in the 50% lethal dose of the mutants over PAO1.

Animals↗

PubMed1990

Restriction fragment length polymorphisms among uropathogenic Escherichia coli isolates: pap-related sequences compared with rrn operons.

Among the adhesin-encoding virulence operons associated with uropathogenic Escherichia coli, only pap (pyelonephritis-associated pilus)-related gene clusters typically exhibit variation in their structure and chromosomal copy number. To access further such variability, we compared pap restriction fragment length polymorphisms (RFLPs) with those detected among rRNA (rrn) operons, which encode an essential host function unrelated to virulence. To place such findings in a phylogenetic perspective, the E. coli isolates were also characterized by using multilocus enzyme electrophoresis. Variation in the rrn RFLP profiles correlated with evolutionary divergence resolved by multilocus enzyme electrophoresis; isolates with identical rrn profiles represented the same or closely related electrophoretic types. In contrast, such isolates frequently had different pap-related RFLPs, indicating that these genetic variations have developed recently relative to the changes associated with essential rrn operons or metabolic enzymes. Despite such fluctuations, two lines of evidence indicate conditions under which the pap-related RFLPs can be stably maintained. First, for each of 20 patients with urosepsis, both the primary urinary tract isolate and the concurrent blood isolate were identical. Second, although obtained from different patients, some isolates representing the same electrophoretic type also had identical pap-related RFLPs. Thus, the genotypic diversity of this virulence adhesin operon was not generated during the course of acute infection or during laboratory manipulations. Since fecal E. coli isolates frequently carry chromosomally encoded pap-related gene clusters, these findings suggest that the intra- and interchromosomal recombination events generating the polymorphisms associated with the pap-related sequences likely occur among the E. coli of the commensal reservoir.

Base Sequence↗

PubMed1993

Cloning and expression of the multiple sugar metabolism (msm) operon of Streptococcus mutans in heterologous streptococcal hosts.

The multiple sugar metabolism (msm) operon of Streptococcus mutans is responsible for the uptake and metabolism of a variety of sugars. In order to further characterize the substrate specificities of the transport system, a 12-kb region of DNA containing the entire msm operon was cloned, via a novel two-step integration strategy, into the chromosomes of two heterologous streptococcal strains, Streptococcus gordonii Challis and Streptococcus anginosus Is57, as well as the chromosome of a natural isolate of S. mutans with a deletion of the msm region. These strains are unable to transport or ferment melibiose, raffinose, or isomaltosaccharides, but the newly constructed recombinants gained the ability to ferment all of these sugars. The S. gordonii Challis construct containing msm was shown to transport radiolabelled melibiose, raffinose, isomaltotriose, and isomaltotetraose, and the transport function was also subjected to induction by raffinose, an inducer of the msm operon in S. mutans. The results confirm the role of the msm operon in the transport and metabolism of melibiose, raffinose, and isomaltosaccharides.

Biological Transport↗

PubMed1996

The pertussis toxin liberation genes of Bordetella pertussis are transcriptionally linked to the pertussis toxin operon.

The DNA sequence of the pertussis toxin operon (ptx) of Bordetella pertussis predicts that transcription of the operon ends downstream from the ptxS3 gene at a possible stem-loop structure. Secretion of the assembled pertussis toxin into the culture medium required the expression of 8 genes arranged in an operon (ptl) and lying 55 bp downstream from the ptx and ptl operons are cotranscribed and coregulated by the P(TOX) promoter. Deletion of the 55-bp DNA region caused an increase in the amount of the ptl transcripts. It is likely that this DNA region is involved in regulation of the ptx-pti expression.

Base Sequence↗

PubMed1997

Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F. tularensis.

The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed. The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins. The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400. The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found. The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress. After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests. When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10. The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.

Antibodies, Bacterial↗

PubMed1999

Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome.

Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen.

Amino Acid Sequence↗

PubMed2000

Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes.

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Abortion, Veterinary↗

PubMed2001

Intracellular induction of the Bartonella henselae virB operon by human endothelial cells.

One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2 gene revealed a region with sequence homology to the vir box of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virB genes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A. tumefaciens.

Bacterial Proteins↗

PubMed2001

Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation.

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> or = 68% similarity) and is preceded by a gene similar to icaR (> or =70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.

Bacteriolysis↗

PubMed2001

Transcriptional regulation of divergent capsule biosynthesis and transport operon promoters in serogroup B Neisseria meningitidis.

The clinically important serogroups B, C, Y, and W-135 of Neisseria meningitidis produce sialic acid capsules that are critical in pathogenesis. In each of these serogroups, the capsule transport (ctrABCD) and capsule biosynthesis (synABCD) operons are divergently transcribed from putative promoters located in a 134-bp intergenic region (J. S. Swartley, J. H. Ahn, L. J. Liu, C. M. Kahler, and D. S. Stephens, J. Bacteriol. 178:4052-4059, 1996). In this study we further assessed the role of the intergenic sequence in the transcriptional regulation of the sialic acid capsules of N. meningitidis. Insertional mutagenesis or deletions of the 134-bp sequence in the serogroup B meningococcal strain NMB resulted in a marked reduction or elimination of ctrABCD and synABCD transcription, with a concomitant loss of encapsulation. Chromosomal transcriptional lacZ-ermC reporter fusions of syn and ctr promoters were constructed through allelic exchange. Using these constructs, both operons were found to be constitutively transcribed in meningococci, the biosynthesis operon about fourfold higher than the transport operon. Both promoters showed increased activity during stationary-phase growth. In addition to the promoters, a 70-bp 5' untranslated region (UTR) upstream of synA was found to have a direct repeat and an inverted repeat that overlapped three putative integration host factor binding sites. Mutation of this 70-bp UTR and of the direct repeat upregulated both syn and ctr transcription. Regulation through the synA UTR was absent in a K1 Escherichia coli strain that produces identical capsular polysaccharide, implicating species-specific regulation. Meningococcal sialic acid capsule expression is initiated by divergent promoters in a 134-bp intergenic region, is repressed at the transcriptional level by the 5' UTR of synA, is increased during stationary-phase growth, and shows species-specific regulation. Transcriptional regulation is another important control point for sialic capsule expression in N. meningitidis.

5' Untranslated Regions↗

PubMed2002

The divergently transcribed Streptococcus parasanguis virulence-associated fimA operon encoding an Mn(2+)-responsive metal transporter and pepO encoding a zinc metallopeptidase are not coordinately regulated.

The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn(2+) concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn(2+). This deficiency was not alleviated by compensation with either Mg(2+) or Fe(3+). Wild-type S. parasanguis could take up Mn(2+) and Fe(3+), while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn(2+) and Fe(3+). FimA expression itself was shown to be responsive to Mn(2+) concentration, but not to availability of Fe(3+) or Mg(2+).

Bacterial Proteins↗