Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.     

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.     

A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly.

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

The anaerobic life of Bacillus subtilis: Cloning of the genes encoding the respiratory nitrate reductase system

Abstract The Gram-positive soil bacterium Bacillus subtilis , generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe. Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth. Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium. The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions. Surprisingly, anaerobically grown B. subtilis lost the capacity for sporulation. An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B. subtilis . Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes. Four open reading frames with significant homology to the E. coli respiratory nitrate reductase opérons ( narGHIJ, narZYWV ) were isolated and termed narGHJI . A chromosomal knock-out mutation of the B. subtilis nar operon totally abolished nitrate respiration.     

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.     

Regulation and symbiotic role of nirK and norC expression in Rhizobium etli

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.     

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.     

Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis.

Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.     

Regulators of aerobic and anaerobic respiration in Bacillus subtilis.

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.