Transport of multidrug resistance substrates by the Streptococcus agalactiae hemolysin transporter

Streptococcus agalactiae (group B streptococcus [GBS]) causes neonatal sepsis, pneumonia, and meningitis, as well as infections of the bovine udder. The S. agalactiae hemolysin is regarded as an important virulence factor, and hemolysin expression is dependent on the cyl gene cluster. cylA and cylB encode the ATP binding and transmembrane domains of a typical ATP binding cassette (ABC) transporter. The deduced proteins contain the signature sequence of a multidrug resistance (MDR) transporter, and mutation of the genes results in a nonhemolytic and nonpigmented phenotype. To further elucidate the function of the putative transporter, nonpolar deletion mutants of cylA were constructed. These mutants are nonhemolytic and can be complemented by the transporter genes. Wild-type strain and nonhemolytic cylA and cylK deletion mutants were exposed to known substrates of MDR transporters. Mutation of cylA significantly impaired growth in the presence of daunorubicin, doxorubicin, and rhodamine 6G and resulted in a decreased export of doxorubicin from the cells. The mutation of cylK, a gene of unknown function located downstream from cylA, caused a loss of hemolysis but had no effect on the transport of MDR substrates. Furthermore, the hemolytic activity of the wild-type strain was inhibited by reserpine in a dose-dependent manner. We conclude that CylAB closely resembles an ABC-type MDR transporter and propose that the GBS hemolysin molecule represents a natural substrate of the transporter.     

Propionibacterium jensenii produces the polyene pigment granadaene and has hemolytic properties similar to those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.     

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.     

Subcellular location and unique secretion of the hemolysin of Serratia marcescens

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.     

Propionibacterium jensenii Produces the Polyene Pigment Granadaene and Has Hemolytic Properties Similar to Those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

Hemolysin as a marker for Serratia

All Serratia marcescens strains (total of 33) of different sources were hemolytic including clinical strains previously classified as being nonhemolytic. DNA fragments of the two hemolysin genes hybridized with the chromosomal DNA of S. marcescens, S. liquefaciens, S. kiliensis, S. grimesii, S. proteamaculans, S. plymutica, S. rubridaea which were also hemolytic. The restriction pattern of the hemolysin locus differed in each strain. S. ficaria and S. marinorubra expressed a different hemolysin which was much smaller than the S. marcescens hemolysin since it diffused through dialysis membranes. The DNA of the latter strains did not hybridize with the S. marcescens hemolysin DNA probes. Some S. marcescens strains, S. kiliensis and S. liquefaciens also expressed in addition the small hemolysin. No hybridization was found with DNA of Escherichia coli, Salmonella typhimurium, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella arerogenes, Klebsiella pneumoniae, Shigella dysenteriae, Yersinia enterocolitica, Yersinia pseudotuberculosus, Listeria sp., Aeromonas sp., Legionella sp. and a Meningococcus sp., indicating that the hemolysin DNA probes are specific for Serratia, or that the hemolysin genes occur rarely in genera other than Serratia.     

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus)

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.     

Transport of hemolysin by Escherichia coli

The hemolytic phenotype in Escherichia coli is determined by four genes. Two (hlyC and hlyA) determine the synthesis of a hemolytically active protein which is transported across the cytoplasmic membrane. The other two genes (hlyBa and hlyBb) encode two proteins which are located in the outer membrane and seem to form a specific transport system for hemolysin across the outer membrane. The primary product of gene hlyA is a protein (protein A) of 106,000 daltons which is nonhemolytic and which is not transported. No signal peptide can be recognized at its N-terminus. In the presence of the hlyC gene product (protein C), the 106,000-dalton protein is processed to the major proteolytic product of 58,000 daltons, which is hemolytically active and is transported across the cytoplasmic membrane. Several other proteolytic fragments of the 106,000-dalton protein are also generated. During the transport of the 58,000-dalton fragment (and possible other proteolytic fragments of hlyA gene product), the C protein remains in the cytoplasm. In the absence of hlyBa and hlyBb the entire hemolytic activity (mainly associated with the 58,000-dalton protein) is located in the periplasm: Studies on the location of hemolysin in hlyBa and hlyBb mutants suggest that the gene product of hlyBa (protein Ba) binds hemolysin and leads it through the outer membrane whereas the gene product of hlyBb (protein Bb) releases hemolysin from the outer membrane. This transport system is specific for E coli hemolysin. Other periplasmic enzymes of E coli and heterologous hemolysin (cereolysin) are not transported.     

Insertional inactivation of streptolysin S expression in Streptococcus pyogenes

The inactivation of a genetic determinant critical for streptolysin S production was accomplished by transfer and insertion of the transposon Tn916 into the DNA of a group A streptococcal strain. The group D strain CG110 was able to efficiently transfer Tn916 into the group A strain CS91 when donor and recipient cells were concentrated and incubated together on membrane filters. Among tetracycline-resistant transconjugants, nonhemolytic mutants that no longer produced streptolysin S and retained the capacity to produce streptolysin O were discovered. Hemolytic revertants from these mutants regained tetracycline sensitivity; other revertants still retained a tetracycline resistance phenotype. Hybridization studies employing Tn916 DNA located Tn916 sequences in EcoRI and HindIII fragments of DNA from mutants devoid of streptolysin S; one carried a single copy of Tn916, and the other two carried multiple copies of the transposon.     

Induction of Yellow Pigmentation in Serratia marcescens

The appearance of yellow pigmentation in nonpigmented strains of Serratia sp. has been demonstrated to be due to the production of a muconic acid, 2-hydroxy-5-carboxymethylmuconic acid semialdehyde. The 3,4-dihydroxyphenylacetate 2,3-dioxygenase responsible for the synthesis of this muconic acid was induced in all strains tested. Another muconic acid, the β-cis-cis-carboxymuconic acid, could also be synthesized from 3,4-dihydroxybenzoate, but this product was not colored. Mutants that were unable to grow on tyrosine and produced yellow pigment were isolated from nonpigmented strains. These mutants had properties similar to those of the yellow-pigmented strains. The ability to produce pigment may be more widespread among Serratia marcescens strains than is currently known.     

Adjacent location of thebac gene and two-component regulatory system genes within the putativeStreptococcus agalactiae pathogenicity island

A chromosomal DNA fragment of 8992 bp in size that has not been previously identified in Streptococcus agalactiae, was cloned and sequenced from strain 98-D60C. In particular, this 8992-bp fragment contained genes homologous to the sensor histidine kinase gene and the DNA-binding response-regulator gene of Streptococcus pneumoniae, and S. agalactiae bac gene. Structural and genetic features of the 8992-bp fragment were highly similar to those specific for bacterial pathogenicity islands. Analysis of epidemiologically unrelated S. agalactiae strains revealed that this fragment was present only in bac gene-positive strains. The possible origin of the 8992-bp fragment in S. agalactiae and its significance for molecular mechanisms of "bacteria-host" interactions are discussed.     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1.

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.     

Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.