hemB Mutant Research Articles

Decreased Susceptibility of Staphylococcus aureus Small-Colony Variants toward Human Antimicrobial Peptides

Staphylococcus aureus is a frequent resident of human nose and skin in many individuals, but it is also able to cause a variety of serious infections including those of the skin and soft tissue. There is increasing evidence that particularly persistent, relapsing, and difficult-to-treat infections caused by S. aureus are associated with the formation of the small-colony variant (SCV) phenotype. The aim of this study was to investigate the hypothesis that (i) skin-derived antimicrobial peptides (AMPs) exhibit a reduced activity against SCVs and (ii) that switching into the SCV phenotype may endow S. aureus with a decreased susceptibility toward the killing activity of human stratum corneum. Here, we show that clinically derived S. aureus SCVs are less susceptible to the bactericidal activity of different human skin-derived AMPs as compared with their isogenic corresponding wild-type strains. Similarly, a S. aureus hemB mutant displaying the SCV phenotype was less susceptible to the antimicrobial activity of AMPs than its hemB-complemented mutant. These findings were accompanied by a higher resistance of SCVs to the killing activity of human stratum corneum. Switching into the SCV phenotype may help S. aureus to subvert cutaneous innate defense, thus contributing to the establishment and persistence of infection.

Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant Staphylococcus aureus are hypersusceptible to -lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species

Phagocytosed methicillin-resistant Staphylococcus aureus (MRSA) are susceptible to β-lactams because of an acid-induced conformational change of penicillin-binding protein (PBP) 2a within phagolysosomes. We have examined whether this mechanism applies to menD and hemB small-colony variants (SCVs) of the COL MRSA strain, using cloxacillin, meropenem, doripenem, and vancomycin as comparator. Intracellularly, the change in cfu from post-phagocytosis inoculum was measured after 24 h of incubation with antibiotics combined or not with N-acetylcysteine (NAC; oxidant species scavenger); the relative potency (C(s)) was calculated from the Hill equation of concentration-response curves. Extracellularly, the effect of a pre-incubation with H(2)O(2) was determined on MICs and killing at pH 7.4 and 5.5. Intracellularly, the β-lactam C(s) was similar for the COL strain and the hemB mutant and not modified or slightly decreased (2- to 16-fold) by NAC. In contrast, the C(s) was 100- to 900-fold lower for the menD mutant, but similar to that for the COL strain when NAC was present. Extracellularly, β-lactam MICs were markedly reduced at pH 5.5 for the parental strain and the haemin-supplemented hemB mutant, with limited additional effect of pre-incubation with H(2)O(2). In contrast, MICs remained elevated at pH 5.5 for the menD mutant (supplemented with menadione sodium bisulphite or not), but were 7-10 dilutions lower after pre-incubation with H(2)O(2). Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on C(s). Cooperation between acidic pH and oxidant species confers high potency to β-lactams against intracellular forms of menD SCVs of MRSA.

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent.

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 106 to 109 colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 107 or 108 CFUs of the normal phenotype, respectively. A minimum dose of 108 or 109 CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm2 vs. 28 mm2). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.

The lipoprotein components of the Isd and Hts transport systems are dispensable for acquisition of heme by Staphylococcus aureus

Heme is a key molecule for Staphylococcus aureus and is involved in many aspects of oxidative metabolism. Crucially, heme is required for the activity of cytochromes of the electron transport chain. Staphylococcus aureus is able to obtain heme either through biosynthesis or through acquisition from the host. Clinically persistent 'small colony variant' (SCV) forms of S.aureus are frequently deficient for heme biosynthesis, and disruption of the hemB gene produces stable heme-auxotrophic strains that reproduce many SCV phenotypes. We sought to address the role of heme transport in SCVs by deleting components of the two described heme import systems, the iron-regulated surface determinant (Isd) and heme transport system (Hts) in wild-type S.aureus and hemB mutant backgrounds. Analysis of the growth of S.aureus hemB strains either singly or doubly deficient in isdE and htsA in the presence and absence of heme or hemoglobin revealed that S.aureus is able to obtain exogenous heme in the absence of these transporter components. These data suggest the presence of additional, as yet unidentified transporter components that enable S.aureus to internalize exogenous heme and contradict the proposed model that IsdE can transfer heme to the HtsBC permease.

Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small-colony variants (SCVs) of Staphylococcus aureus represent a slow-growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild-type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin-induced SCV). Applying an ultra-high resolution chromatography and high mass accuracy MS(E) -based label-free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase-controlled manner covering early-exponential, late-exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down-regulation of the tricarboxylic acid (TCA) cycle-related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin-induced SCVs, the clinically derived SCVs showed no prominent up-regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up-regulation of TCA cycle-related parts, while oxidative stress-related proteins were down-regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain-interrupting mutants and their complemented counterparts.

Extracellular and intracellular bactericidal activities of XF-70 against small-colony variant hemB mutants of meticillin-susceptible and meticillin-resistant Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus are phenotypic variants characterised by their small colony size and improved intracellular survival and are associated with persistent and relapsing infections. XF drugs are membrane-active, porphyrin-based antibacterial agents for topical administration, exerting rapid bactericidal activity against actively growing or resting, antibiotic-susceptible and multidrug-resistant strains of S. aureus. In this study, minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of XF-70 against isogenic, electron-transport deficient, SCV hemB mutants of one meticillin-susceptible S. aureus (MSSA) strain and one meticillin-resistant S. aureus (MRSA) strain were evaluated. Macrodilution MICs of XF-70 for MSSA strain 8325-4 and its hemB +-complemented derivative (0.5–1 mg/L) were reproducible and were slightly higher than that for the SCV hemB mutant (0.25–0.5 mg/L) and were not influenced by increasing inoculum size from 10 6 to 10 8 colony-forming units (CFU)/mL. MICs for MRSA strain COL, its SCV hemB mutant and hemB +-complemented derivative were equivalent (0.25–1 mg/L). MBCs of XF-70 were ≤2-fold higher than MICs for all isolates. Extensive killing (≥4 log reduction in CFU/mL) was produced by 2 mg/L XF-70 within 30 min against SCV hemB mutants both of 8325-4 and COL as well as their respective parent or hemB +-complemented derivatives. Pre-incubation of 10 7 CFU/mL of 8325-4 and its SCV hemB mutant with 5 × 10 6 polymorphonuclear neutrophils for 30 min markedly protected phagocytised organisms from rapid extensive killing by bactericidal levels (2 mg/L) of subsequently added XF-70. The rapid bactericidal activity of XF-70 at low concentrations both against SCV and normally growing S. aureus is remarkable and represents an attractive potential for the treatment of persistent localised infections.

Staphylococcus aureusSmall‐Colony Variants Are Adapted Phenotypes for Intracellular Persistence

Staphylococcus aureus is an important human pathogen of endovascular diseases, which can take a chronic course with a high relapse rate despite antimicrobial treatment. Thus far, persistent and antibiotic-refractory infections have been largely associated with a subpopulation of S. aureus, the small-colony variants (SCVs). In this work, we used endothelial cells to investigate infection with the highly virulent wild-type isolate (6850), 2 stable isogenic SCV phenotypes (hemB mutant IIb13 and JB1), and the complemented mutant. All strains were highly invasive in endothelial cells but largely differed in host response induction. Microarray analysis showed that wild-type phenotypes up-regulated a large number of endothelial genes (including genes involved in innate immunity), whereas the SCVs did not cause these dramatic changes. The inflammatory response and cytotoxicity were strongest shortly after infection and largely decreased within the following days, which was accompanied by a fast elimination of intracellular wild-type bacteria. By contrast, SCVs survived within endothelial cells at high numbers. S. aureus intracellular persistence via the development of an adapted subpopulation of SCVs most likely represents an important strategy of S. aureus to hide within the host cells, which could be a reservoir for chronic infections.

Antibody and cell-mediated immune responses to Staphylococcus aureus small colony variants and their parental strains associated with bovine mastitis

Persistence of bovine Staphylococcus aureus mastitis may be associated with the small colony variant (SCV) form that is adapted to intracellular life and resists elimination by the immune system. This study evaluated antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) to two bovine SCV forms and their parent strains isolated from cows with mastitis. Four groups of healthy cows, five cows/treatment group, were challenged by the intramammary route with naturally occurring bovine SCV Heba3231, its parent strain 3231, a hemB mutant displaying the SCV phenotype or its parent strain, Newbould 305. Blood and milk samples were collected at day 0 before challenge and at days 1, 14, 21 and 36 post-challenge to determine antigen-specific immunoglobulin (Ig) IgG 1 and IgG 2 antibody responses as indicators of type 2 and type 1 responses, respectively. At day 24 post-challenge cows in each group were inoculated with the UV-killed homologous strain intradermally in the neck to induce delayed-type hypersensitivity (DTH) as an indicator of CMIR. The SCV Heba3231 and 3231 strains induced significant IgG 1 and IgG 2 antibody responses in sera and in sera and milk whey, respectively. The hemB SCV mutant and Newbould 305 strains induced significant IgG 1 antibody in milk whey, and in sera and milk whey, respectively. The SCV Heba3231 and 3231 strains induced DTH, the hemB mutant induced intermediate hypersensitivity, and Newbould 305 failed to induce DTH. These results indicate marked differences in immune responses induced by parent and SCV forms of the same strain of S. aureus and by the two wild-type strains. This is the first study to evaluate both AMIR and CMIR in the context of persistent bovine mastitis to different and genetically characterized strains of S. aureus including two SCVs. The findings expand our understanding of immune responses to persistent S. aureus mastitis.

Daptomycin pharmacodynamics against Staphylococcus aureus hemB mutants displaying the small colony variant phenotype

Staphylococcus aureus small colony variants (SCVs) are slow-growing morphological variants associated with persistent infections. While vancomycin activity has been shown to be attenuated against SCVs of S. aureus, few data exist regarding daptomycin. The objective was to evaluate the pharmacodynamics of daptomycin against defined S. aureus mutants displaying the SCV phenotype. Two S. aureus hemB mutants (Ia48 and III33) displaying the SCV phenotype and their parental strains (COL and Newman) were evaluated. Time-kill experiments were performed using a starting inoculum of 10(6) cfu/mL at 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 times the MIC. Samples were obtained at 0, 1, 2, 4, 6, 8 and 24 h, plated and incubated to determine colony counts. A Hill-type pharmacodynamic mathematical model was fitted to the data to characterize the effect. Bactericidal activity for daptomycin was achieved and occurred in a concentration-dependent manner against both hemB mutants and their parental strains. Against strains with normal phenotype, bactericidal activity was achieved rapidly, within 2 h at concentrations > or =16 times the MIC, while against SCVs, bactericidal activity was achieved within 6 h at concentrations > or =16 times the MIC. Against both hemB mutants, daptomycin maintained bactericidal activity at 24 h, with similar profiles of killing activity when compared with their parental strains. Daptomycin achieved bactericidal activity against S. aureus hemB mutants and parenteral isolates. Daptomycin represents a potential therapeutic option for infections caused by S. aureus strains displaying the SCV phenotype and additional studies are warranted.

Somatic cell scores and clinical signs following experimental intramammary infection of dairy cows with a Staphylococcus aureus small colony variant ( S. aureus SCV) in comparison to other bovine strains

A recently isolated bovine Staphylococcus aureus small colony variant (SCV) was shown to persist within cultured endothelial cells. However, the clinical importance of S. aureus SCV in persistent infection of the mammary gland is unknown. The hypothesis tested here was a naturally occurring bovine S. aureus SCV, Heba3231, and a Newbould hemB mutant that was an artificially created SCV would establish mild intramammary infection and induce a different host response compared to their isogenic parental strains, 3231 and Newbould 305. Four groups of clinically healthy cows, 5 cows/treatment group, were infected by the intramammary route with SCV Heba3231, strain 3231, the hemB mutant or strain Newbould 305. Three quarters of the mammary gland of each cow were challenged with ∼5000 colony-forming units of bacteria and the fourth quarter was infused with PBS. Cows were monitored and assigned clinical scores for the first 5 days post-challenge based on rectal temperature, appetite, milk yield, udder uniformity, milk appearance, bacterial culture of foremilk samples and somatic cell score (SCS). All cows were examined for chronic infection up to day 36 post-challenge. Cows challenged with SCV strains developed mastitis that was mild compared with that induced by the parent strains. Cows challenged with strain 3231 developed the most severe clinical mastitis. SCS from all treatment groups were significantly ( P < 0.05) higher at days 2–5 and 14–36 post-infection compared to day 0 before challenge. This milder host response to SCVs may represent an adaptation of S. aureus to the mammary gland environment.

Comment on: High tigecycline resistance in multidrug-resistant Acinetobacter baumannii

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Staphylococcus aureus small colony variants are resistant to the antimicrobial peptide lactoferricin B

To determine whether Staphylococcus aureus small colony variants (SCVs) are resistant to the antimicrobial peptide lactoferricin B. To assess if deficiency in transmembrane potential, a common characteristic of SCVs that are haemin- or menadione-auxotrophs, affects the uptake of the peptide into the bacterial cytoplasm. A broth microdilution technique was used for susceptibility testing to determine the MIC of lactoferricin B for SCVs with three different auxotrophisms (haemin, menadione or thymidine) and their isogenic parent strains. Both clinical isolates and genetically defined mutants were used. The internalization of lactoferricin B in a hemB mutant and the respective parent strain was studied using transmission electron microscopy and immunogold labelling. All SCVs showed reduced susceptibility to lactoferricin B irrespective of their auxotrophy compared with their isogenic parent strains. The MIC for all SCVs was >256 mg/L, whereas the MICs for the parent strains ranged from 16-256 mg/L. Surprisingly, the hemB mutant contained significantly more lactoferricin B intracellularly than the respective parent strain. The resistance mechanism of SCVs towards the antimicrobial peptide lactoferricin B is presumably caused by the metabolic changes present in SCVs rather than by a changed transmembrane potential of SCVs or reduced uptake of the peptide.

SigmaB activity in a Staphylococcus aureus hemB mutant.

Inactivation of hemB in Staphylococcus aureus strain Newman resulted in a small-colony phenotype and was accompanied by an altered expression pattern of global regulators and control of virulence factor production. Transcription profiles followed over 15 h by Northern blot analyses revealed that transcripts of the global regulators arl, rot, sae, sarR, sarS, srr, svrA, and sigB disappeared after the exponential phase and that both agr transcripts were completely absent in the hemB mutant. Apart from a general concentration of transcriptional activity to the exponential phase, premature gene expression was observed for rot, hla, and spa. Nevertheless, reported sigmaB-dependent transcripts, such as sarC and clfA, were produced throughout the 15-h growth period monitored. The absence of these transcripts in a hemB sigB double mutant demonstrated their dependence on sigmaB and indicated an unexpected, permanent sigmaB activity in the hemB mutant. Variations in the extents of the directly sigmaB-controlled asp23, rsbVW-sigB, and sarC transcripts argue for additional factors modulating sigmaB activity. This study provides the first extended synopsis of the transcriptional patterns of different regulators over the entire growth cycle in the widely used Newman strain.

Persistence of a Staphylococcus aureus small-colony variant under antibiotic pressure in vivo

Staphylococcus aureus small-colony variants (SCVs) have been implicated in chronic and persistent infections. Bovine mastitis induced by S. aureus is an example of an infection difficult to eradicate by conventional antimicrobial therapies. In this study, the ability to colonize mouse mammary glands and persist under antibiotic treatment was assessed for S. aureus Newbould and an isogenic hemB mutant, which exhibited the classical SCV phenotype. The hemB mutant showed a markedly reduced capacity to colonize tissues. However, although the hemB mutant was as susceptible as S. aureus Newbould to cephapirin in vitro, it was over a 100 times more persistent than the parental strain in the mammary glands when 1 or 2 mg kg −1 doses were administrated. These results suggest that, although the hemB mutant has a reduced ability to colonize mammary glands, the SCV phenotype may account for the persistence of S. aureus under antibiotic pressure in vivo.

GP003 Identification of an agr homologue in Staphylococcus saprophyticus

Staphylococcus aureus is a frequent resident of human nose and skin in many individuals, but it is also able to cause a variety of serious infections including those of the skin and soft tissue. There is increasing evidence that particularly persistent, relapsing, and difficult-to-treat infections caused by S. aureus are associated with the formation of the small-colony variant (SCV) phenotype. The aim of this study was to investigate the hypothesis that (i) skin-derived antimicrobial peptides (AMPs) exhibit a reduced activity against SCVs and (ii) that switching into the SCV phenotype may endow S. aureus with a decreased susceptibility toward the killing activity of human stratum corneum. Here, we show that clinically derived S. aureus SCVs are less susceptible to the bactericidal activity of different human skin-derived AMPs as compared with their isogenic corresponding wild-type strains. Similarly, a S. aureus hemB mutant displaying the SCV phenotype was less susceptible to the antimicrobial activity of AMPs than its hemB-complemented mutant. These findings were accompanied by a higher resistance of SCVs to the killing activity of human stratum corneum. Switching into the SCV phenotype may help S. aureus to subvert cutaneous innate defense, thus contributing to the establishment and persistence of infection.

Staphylococcus aureus menD and hemB mutants are as infective as the parent strains, but the menadione biosynthetic mutant persists within the kidney.

Small colony variants (SCVs) of Staphylococcus aureus were generated via mutations in menD or hemB, yielding menadione and hemin auxotrophs, respectively, and studied in the rabbit endocarditis model. No differences in the 95% infectious dose occurred between strains with regard to seeding heart valves ( approximately 10(6) cfu) or other target organs. No differences were observed between the response of the hemB mutant to oxacillin therapy and that of the parent strain in any target tissues, and significant reductions in bacterial densities were seen in all tissues (compared with untreated controls). In contrast, oxacillin therapy did not significantly reduce bacterial densities of the menD mutant in either kidney or spleen and significantly reduced densities within vegetations. These data show that SCVs are able to colonize multiple tissues in vivo and that the menD mutation provides the organism with a survival advantage during antimicrobial therapy, compared with its parent strain, in selected target tissues.

Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis

Persistence of Staphylococcus aureus during invasive infections has been associated with a small-colony variant (SCV) phenotype. SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain. SCVs have been shown to be more resistant to antibiotics such as aminoglycosides, grow slowly and persist intracellularly. The aim of this study was to assess the virulence of an hemB mutant, which has been shown to display the typical characteristics of clinical SCVs, in a murine model of septic arthritis. NMRI mice were inoculated intravenously with either the wild type strain Newman or with its mutant displaying the SCV phenotype. The clinical, bacteriological, and histopathological progression of the disease was studied. Mice inoculated with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the wild type Newman strain. Despite that, the mutant inoculated mice displayed significantly lower bacterial burden in their kidneys and joints compared with mice exposed to the wild parental strain. Notably, the hemB mutant produced almost 20 times more protease in vitro than the parental strain. We conclude that the small colony variants of S. aureus are more virulent on a per organism basis than its isogenic parental strain in the model of septic arthritis. This can at least in part be explained by the ability of SCV to produce high amounts of destructive proteases.

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of α-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants.

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of alpha-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase.

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.