Phenotype Microarray Profiling of Staphylococcus aureus menD and hemB Mutants with the Small-Colony-Variant Phenotype

Abstract

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus smallcolony 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.

Fourier-Transform Infrared Spectroscopic Analysis Is a Powerful Tool for Studying the Dynamic Changes in Staphylococcus aureus Small-Colony Variants

“…On solid media, the unique SCV phenotype may be recognized visually if cells grow unambiguously as pinpoint colonies. However, naturally occurring SCVs are unstable, and most studies on the SCV phenotype are based upon stable site-directed mutants in electron transport (2,19,23,26,27). Because the mutations in clinical strains have yet to be defined, these mutants do not necessary reflect the complete phenotype of natural SCVs.…”

Regulatory Overlap and Functional Redundancy amongBacillus subtilisExtracytoplasmic Function σ Factors

“…Phenotype microarrays allow the analysis of hundreds of physiological tests in parallel, thereby enabling the fast and accurate identification of novel traits linked to genetic alterations (1,34,40). Remarkably, a comparison of the quadruple mutant HB0842 with the isogenic wild type revealed only three significant differences in growth: the mutant grew better than wild type in the presence of chloramphenicol, neomycin, and piperacillin.…”

Advantage of Upregulation of Succinate Dehydrogenase in Staphylococcus aureus Biofilms

“…Genetically stable mutants in the hemin biosynthetic gene, hemB, or menadione biosynthetic gene, menD, have been characterized as having lower capacities for oxidative phosphorylation. They revealed many phenotypic changes (e.g., decreased pigmentation) and unusual biochemical characteristics as consequences of altered metabolic and energy-dependent pathways (4,5,21,38,46,48,49).In a previous study, it was found that some TCA cycle genes were upregulated in biofilms, particularly the succinate dehydrogenase genes (sdhCAB) but also the 2-oxoglutarate dehydrogenase (odhA) and the succinyl-coenzyme A (CoA) synthetase (sucC) genes (32, 33). The succinate dehydrogenase (EC 1.3.99.1) is part of the nonoxidative branch of the TCA cycle and is directly linked to the respiratory chain.…”

“…Genetically stable mutants in the hemin biosynthetic gene, hemB, or menadione biosynthetic gene, menD, have been characterized as having lower capacities for oxidative phosphorylation. They revealed many phenotypic changes (e.g., decreased pigmentation) and unusual biochemical characteristics as consequences of altered metabolic and energy-dependent pathways (4,5,21,38,46,48,49).…”