Brigitte Berger-Bächi

Microarray-Based Analysis of the Staphylococcus aureus σB Regulon

Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by σB activity. While σB was found to positively control 198 genes by a factor of ≥2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of σB. Gene products that were found to be influenced by σB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by σB were preceded by a nucleotide sequence that resembled the σB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by σB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the σB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.

Factors influencing methicillin resistance in staphylococci

Abstract. Methicillin resistance in staphylococci is due to an acquired penicillin-binding protein, PBP2′ (PBP2a). This additional PBP, encoded by mecA, confers an intrinsic resistance to all β-lactams and their derivatives. Resistance levels in methicillin-resistant Staphylococcus aureus (MRSA) depend on efficient PBP2′ production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired mecA, resistance levels range from phenotypically susceptible to highly resistant. Characteristic for most MRSA is the heterogeneous expression of resistance, which is due to the segregation of a more highly resistant subpopulation upon challenge with methicillin. Maximal expression of resistance by PBP2′ requires the efficient and correct synthesis of the peptidoglycan precursor. Genes involved in cell-wall precursor formation and turnover, regulation, transport, and signal transduction may determine the level of resistance that is expressed. At this stage, however, there is no information available on the functionality or efficacy of such factors in clinical isolates in relation to methicillin resistance levels.

FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization.

SummaryThe methicillin resistance determinant (mec) in Staphylococcus aureus resides on additional DNA not present in isogenic sensitive cells. However, besides mec, other chromosomally determined factors are essential for expression of methicillin resistance. We cloned and characterized a chromosomally determined gene which encodes a factor essential for the expression of methicillin resistance (femA) in S. aureus. femA mapped in chromosomal segment number 18, genetically very distant from the methicillin resistance determinant (mec). The product of femA was a protein of an apparent size of 48 kDa. FemA restored methicillin resistance in S. aureus that had become sensitive to methicillin by insertion of Ω22003 (femA::Tn551). Although FemA was needed for cell growth in the presence of β-lactam antibiotics, it had no influence on the synthesis of the low affinity, additional penicillin-binding protein (PBP2′) encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin. Nucleotide sequence analysis, Northern RNA blotting and S1 nuclease RNA mapping suggested that femA was transcribed on a polycistronic mRNA. This mRNA contained the coding region for FemA (ORF433) and a second coding region (ORF419) producing a protein of 47 kDa. The nucleotide and amino acid sequence of FemA showed homologies with ORF419, suggesting that these genes arose by gene duplication. In addition we present evidence for a second chromosomal factor, femB, involved in expression of methicillin resistance which maps close to femA.

Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus

Staphylococcus aureus peptidoglycan is cross‐linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t‐RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP‐MurNAc‐pentapeptide, lipid I and lipid II and the staphylococcal t‐RNA pool, as well as His‐tagged Gly‐tRNA‐synthetase and His‐tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II‐Gly1 and lipid II‐Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane‐associated steps of S. aureus peptidoglycan biosynthesis.

Fitness Cost of SCCmec and Methicillin Resistance Levels in Staphylococcus aureus

ABSTRACT Transformation of a type I SCCmec element into Staphylococcus aureus yielded highly oxacillin-resistant transformants with a reduced growth rate. Faster-growing variants could again be selected at the cost of reduced resistance levels, demonstrating an inverse correlation between oxacillin resistance levels and growth rate.

Mechanisms of heteroresistance in methicillin-resistant Staphylococcus aureus.

Characteristic for methicillin-resistant (Mcr) staphylococci is the heterogeneous expression of the intrinsic methicillin resistance. The majority of the cells express resistance to low concentrations of methicillin, and a minority of the cells express resistance to much higher concentrations. We show here (i) that the presence of the mecA encoding region on plasmid pBBB79 was sufficient to render a methicillin-susceptible (Mcs) Staphylococcus aureus strain heteroresistant and (ii) that this Mcr strain segregated highly resistant subclones which retained the high-resistance phenotype under nonselective growth conditions. The Mcr strain with only mecA on plasmid pBBB79 thus behaved identically to a Mcr strain carrying the complete mec determinant integrated at its proper chromosomal site. (iii) Curing a such highly resistant subclone from plasmid pBBB79 yielded an Mcs strain that was as susceptible as the original Mcs parent strain. (iv) Comparisons were made between the original parent and the cured Mcs strain by backcrossing pBBB79 into them and looking at their progeny. Transductants derived from the formerly highly resistant cured strain became resistant to high concentrations of methicillin, whereas transductants derived from the original parent strain were resistant to lower concentrations of methicillin and showed the typical heterogeneous resistance. We deduced therefrom that the high-level resistance expressed by the minority of the population of Mcr S. aureus was due to a chromosomal mutation(s) (chr*) involving neither mecA nor the additional 30 kb of mec-associated DNA. Moreover, we could show that this postulated mutation chr* was not linked to the femAB operon, which is known to affect methicillin resistance levels.

Fitness Cost of Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus aureus by Way of Continuous Culture

ABSTRACT We examined the effect of introducing type I or IV staphylococcal cassette chromosome mec (SCCmec) elements on the growth yield of Staphylococcus aureus in glucose-limited continuous culture. Type I showed increased glucose consumption and ATP demand per gram of cells synthesized and decreased cell yield compared to those of the parent strain. In contrast, type IV SCCmec elements had no adverse energetic effect.

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

BackgroundThe catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.ResultsThe addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.ConclusionThis study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

Staphylococcus aureus CcpA affects biofilm formation.

ABSTRACT Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA, coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans-Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus (arlRS, mgrA, rbf, sarA, atl, ica, citZ, citB, and cidABC) showed that CcpA up-regulated the transcription of cidA, which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ.