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A large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-long Streptococcus pyogenes serotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces the ori site relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, both in vitro and in vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place near oriC always happen to secure the linkage of oriC to DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed. IMPORTANCE Based on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of the S. pyogenes genome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growth in vitro and in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of the oriC and the chromosome partition cluster is preserved.

A large-scale genomic inversion encompassing 0.79 Mb of the 1.816 Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. As compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in exponential phase. Similarly, when cultured separately in rich medium during prolong stationary phase or in an experimental acute infection animal model (Galleria mellonella), the survival rate of both the parental strain and the invertant is equivalent. However, when co-incubated together, both in vitro and in vivo, the survival of the invertant declines relative to the parental strain. The accompanying aspect of the study suggests that inversions taking place nearbyoriC, always happen to secure the linkage oforiC to DNA sequences responsible for chromosome partition. The biological relevance of large scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb vs 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicate that even large genomic rearrangements survive provided that the integrity of theoriC and the chromosome partition cluster is preserved.

Streptolysin O (SLO) is a member of the thiol-activated cytolysins found in a number of Gram positive pathogenic bacteria. Often accompanying these cytolysins is a second gene first identified in Clostridium perfringens as the putative perfringolysin O regulator (pfoR). Members of the pfoR family are integral membrane proteins and exclusively found in exotoxin producing low %G+C Gram positive bacteria. The homolog of pfoR found in Streptococcus pyogenes is gene Spy0146 or slo R, a member of a three gene operon. In contrast to all other species, slo R is co-transcribed with another gene, Spy0145, a member of a large family of predicted transcriptional regulators of the yjg F family. The third gene (Spy0144) is a predicted DNA binding protein that may function as a regulator for the operon. We previously had shown that inactivation of SloR modulates the expression of SLO expression. To examine the effect of the slo R operon on SLO hemolysis, S. pyogenes strain NZ131 or its isogenic mutants for slo R (OK85) or Spy0144 (NZ131ΔSpy144) were grown in rich medium, with supernatant samples harvested between early-logarithmic and stationary phases. Trypan blue was used to inhibit SLS activity. Hemolysis in strain OK85 ( slo R-) was delayed as compared to wild type by approximately one cell division, while strain NZ131ΔSpy0144 showed an early expression of hemolysis as compared to wild type. The inactivation of Spy0144 appears to be related to increased expression of slo R and up-regulation of SLO. Restoration of a functional copy of Spy0144 on a bacteriophage integration vector was able to partially restore the wild type phenotype. The findings suggest that the slo R operon may encode an environmental sensor that relays some external signal to the cell, modulating the transcription of SLO through a yet to be determined molecular pathway.

The complete genome sequence of nine different strains of Streptococcus pyogenes have now been determined, including serotype M1 and M3 (two strains each), M5, M6, M18, M28, and M49. Seven of these strains are included in the class I type, known to be associated with either acute rheumatic fever or an invasive disease. Class II organisms are known to be associated with acute glomerulonephritis (AGN) and one of the best studied of this class is the M49 strain NZ131, the complete sequence of which has now been determined. The NZ131 (M49) genome contains 1,815,858 bp and is essentially colinear with the M1 genome; it contains no inversions around the origin or terminus. NZ131 contains several large blocks of DNA that appear to be insertions when compared to the class I genomes. These blocks do not appear to be mobile genetic elements, range in size from 3 to 28 kb, and contain a number of genes not found in any of the other sequenced strains. It is noteworthy that this genome contains only two complete bacteriophages—the least number of phages present in any of the previously described genomes. Interestingly, one of these phage genomes is inserted into the same location on the M49 genome as phage 370.2 on the M1 genome. Additionally, this phage contains a single superantigen gene (speH) at the attachment site, but not both speH and speI. A second phage genome contains a streptodornase gene at its attachment site. It is now becoming clear that homologous recombination between various prophages which share homology and residing in the same host can lead to host genome rearrangements and new associations with lysogenic conversion genes in phages. Further analysis of the unique M49 genes should provide important new information regarding virulence genes involved in AGN.

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor inEscherichia colicells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. ThehslJgene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium.cysBmutants showed upregulation of thehslJ : : lacZgene fusion and exhibited increased novobiocin resistance. In this study thehslJtranscription start point and the corresponding putativeσ70promoter were determined. ThehslJpromoter region was defined by employing differenthslJ–lacZoperon fusions, and transcription of thehslJgene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor ofhslJtranscription and binds thehslJpromoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligandN-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression ofhslJtranscription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.

The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production.

In a previous study, we demonstrated the existence of a gene locus, nov, which affects resistance of Escherichia coli K-12 to the gyrase inhibitor novobiocin and, to a lesser degree, coumermycin (J. Rakonjac, M. Milic, D. Ajdic, D. Santos, R. Ivanisevic, and D. J. Savic, Mol. Microbiol. 6:1547-1553, 1992). In the present study, sequencing of the nov gene locus revealed one open reading frame that encodes a protein of 54,574 Da, a value. found to be in correspondence with the size of the Nov protein identified in an in vitro translation system. We also located the 5' end of the nov transcript 8 bp downstream from a classical sigma70 promoter. Transcription of the gene is in the counterclockwise direction on the E. coli chromosome. Transposon mutagenesis of nov followed by complementation analyses and replacement of chromosomal alleles with mutated nov confirmed our previous assumption that the nov gene exists in two allelic forms and that the Novr gene is an active allele while the Novs gene is an inactive form. After comparing nucleotide sequences flanking the nov gene with existing data, we conclude that the gene order in this region of the E. coli K-12 map is att phi 80-open reading frame of unknown function-kch (potassium channel protein)-nov-opp. Finally, the possible identity of the nov gene with cls, the gene that codes for cardiolipin synthase, is also discussed.

The mechanisms that allow Streptococcus pyogenes to survive and persist in the human host, often in spite of antibiotic therapy, remain poorly characterized. Therefore, the determination of culture conditions for long-term studies is crucial to advancement in this field. Stationary cultures of S. pyogenes strain NZ131 and its spontaneous small-colony variant OK171 were found to survive in rich medium for less than 2 weeks, and this inability to survive resulted from the acidification of the medium to below pH 5.5, which the cells did not tolerate for longer than 6–7 days. The growth of NZ131 resulted in acidification of the culture to below pH 5.5 by the onset of stationary phase, and the loss of viability occurred in a linear fashion. These results were also found to be true for M49 strain CS101 and for M1 strain SF370. The S. pyogenes strains could be protected from killing by the addition of a buffer that stabilized the pH of the medium at pH 6.5, ensuring bacterial survival to at least 70 days. By contrast, increasing the glucose added to the medium accelerated the loss of culture viability in strain NZ131 but not OK171, suggesting that the small-colony variant is altered in glucose uptake or metabolism. Similarly, acidification of the medium prior to inoculation or at the middle of exponential phase resulted in growth inhibition of all strains. These results suggest that control of the pH is crucial for establishing long-term cultures of S. pyogenes.

Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Novr) mutants: 10% of Novr mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.

The 1,815,783-bp genome of a serotype M49 strain ofStreptococcus pyogenes(group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3;emmpattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequencedS. pyogenesgenomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-likeemmregion that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49S. pyogenesstrains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.

Escherichia coli possesses three well-established DNA polymerases, I, II, and III. DNA polymerase I (Pol I) is the main repair polymerase in E. coli and also has a minor but important role in chromosomal replication. A major advantage of Pol I as an experimental system is its simplicity; unlike other replication enzymes, it is active as a single subunit. To a large extent, mutagenesis appears to be the result of (dis)functions of the DNA replication machinery. It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events.

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O ( slo ) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.

We report on a significant difference between the published nucleotide sequence of the Escherichia coli K12 histidine operon and our sequencing results repeatedly obtained from a number of different E. coli K12 strains. The discrepancies include 39 base-pair changes and one addition located predominantly in the proximal portion of the operon. Our data also suggest that neutral and near-neutral mutations do not accumulate to a significant extent in the histidine operon of E. coli strains harbouring strong mutator alleles.

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes , a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial “molecular mimicry” of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

Summary We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between atto80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.

The sources of spontaneous mutations and molecular details that underlay the production of spontaneous mutations are still poorly understood. One source is related to DNA metabolism involving DNA replication and repair (1,2). Some of the mutator loci indentified in E. coli are related to structural genes for DNA polymerase, and one of them is related to the polA gene, the gene for DNA polymerase I.

Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150,000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5'-GCTGGCTGGCTGGCTG-3'. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong mutator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5'-GTGG-3' sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.

In this work, the previously described inversion in the lactose region of the Escherichia coli K-12 chromosome (D. J. Savić, J. Bacteriol. 140:311-319, 1979) is analyzed in greater detail. The results presented indicate that the inversion most likely occurred by a homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements.

An unlinked regulatory mutation hisT1504, causes an approximate 11-fold derepression of the histidine (his) operon and a linked constitutive mutation hisO1242 causes an approximate 15-fold derepression. In this study we demonstrate that hisT1504 provokes a significant increase in the UV-induced reversion frequency of his ochre and frameshift mutations. Analysis of revertants derived from frameshift mutants show that this increment in derepressed strains compared to the repressed strains is due to better growth of suppressed revertants by weak frameshift suppressors. The frequency of revertants suppressed by strong frameshift suppressors appears to be the same in repressed and derepressed strains. In contrast, intragenic revertants appear at two-fold decreased frequency in derepressed strains carrying either of the histidine constitutive mutations, hisT1504 or hisO1242. A possible competition is indicated between frequently transcribing RNA polymerase and error-promoting recombinational repair within the histidine operon.

The presence of the histidine operator-constitutive mutationhis01242 increases UV-induced mutability within the histidine operon ofSalmonella typhimurium. The rate of reversion ofhisC andhisF ochre and frameshift mutants is increased 5- to 8-fold when these mutations are coupled withhis01242 which causes 15-fold derepression of the operon. The effect does not extend to the whole chromosome since the rate of UV-induced mutability at the unlinked streptomycin locus is the same in the strains carryinghis0+ orhis01242 alleles. The same phenomenon was observed in Hcr− strains.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.

A constitutive mutation in the histidine operon in Salmonella leads to a decrease in the recombinant recovery following transduction with P22 phage. This decrease appears to be a true decrease in recombination within the histidine operon and specific to that gene region. In addition, a strain with an unlinked mutation was isolated in which an operator-constitutive mutation is lethal.

This paper presents the first detailed structural analysis of termini of an inversion mediated by recombination between Escherichia coli native IS elements. The complete nucleotide sequence of the inversion termini in the lactose region of Escherichia coli K-12 confirms our previous suggestion that the inversion occurred by homologous recombination between alpha 3 beta 3 and beta 5 alpha 5 IS3 elements (D. J. Savic, J. Bacteriol. 140:311-319, 1979; D. J. Savic, S. Romac, and S. D. Ehrlich, J. Bacteriol. 155:943-946, 1983). The data show a slight structural divergence of alpha 3 beta 3 and beta 5 alpha 5 elements, but they do not reveal new sequences within recombined IS3 elements that could influence the expression of nearby genes.