Transcriptomic response of Listeria monocytogenes to iron limitation and Fur mutation.

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.

Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus.

Heat-shock proteins are essential for stress tolerance and allowing organisms to survive conditions that cause protein unfolding. The role of the Staphylococcus aureus DnaK system in tolerance of various stresses was studied by disruption of dnaK by partial deletion and insertion of a kanamycin gene cassette. Deletion of dnaK in S. aureus strain COL resulted in poor growth at temperatures of 37 degrees C and above, and reduced carotenoid production. The mutant strain also exhibited increased susceptibility to oxidative and cell-wall-active antibiotic stress conditions. In addition, the mutant strain had slower rates of autolysis, suggesting a correlation between DnaK and functional expression of staphylococcal autolysins. Deletion of dnaK also resulted in a decrease in the ability of the organism to survive in a mouse host during a systemic infection. In summary, the DnaK system in S. aureus plays a significant role in the survival of S. aureus under various stress conditions.

Molecular characterization of the Fur protein of Listeria monocytogenes.

Iron is essential for the survival of almost all organisms, although excess iron can result in the generation of free radicals which are toxic to cells. To avoid the toxic effects of free radicals, the concentration of intracellular iron is generally regulated by the ferric uptake regulator Fur in bacteria. The 150 aa fur ORF from Listeria monocytogenes was cloned into pRSETa, and the His-tagged fusion protein was purified by nickel affinity column chromatography. DNA binding activity of this protein was studied by an electrophoretic mobility shift assay using the end-labelled promoters P(fhuDC) and P(fur). The results showed a decrease in migration for both promoter DNAs in the presence of the Fur protein, and the change in migration was competitively inhibited with an excess of the same unlabelled promoters. No shift in migration was observed when a similar assay was performed using non-specific end-labelled DNA. The assay showed that binding of Fur to P(fur) or P(fhuDC) was independent of iron or manganese ions, and was not inhibited in the presence of 2 mM EDTA. Inductively coupled plasma MS of the Fur protein showed no iron or manganese, but 0.48 mole zinc per mole protein was detected. A DNase I protection assay revealed that Fur specifically bound to and protected a 19 bp consensus Fur box sequence located in the promoters of fur and fhuDC. There was no requirement for iron or manganese in this assay also. However, Northern blot analysis showed an increase in fur transcription under iron-restricted compared to high-level conditions. Thus, the study suggests that under in vitro conditions, the affinity of the Fur protein for the 19 bp Fur box sequence does not require iron, but iron availability regulates fur transcription in vivo. Thus, the regulation by Fur in this intracellular pathogen may be dependent on either the structure of the DNA binding domain or other intracellular factors yet to be identified.

Autolytic properties of glycopeptide-intermediate Staphylococcus aureus Mu50.

Whole-cell autolytic activity of prototypical glycopeptide-intermediate Staphylococcus aureus (GISA) Mu50 was reduced versus that of hetero-GISA Mu3 and glycopeptide-susceptible S. aureus, consistent with other GISA strains. In contrast, autolytic activity was relatively high in Mu50 crude cell walls and autolysin extracts against purified cell walls, reflecting the complexities of autolytic activity regulation.

Characterization of a multicopper oxidase gene from Staphylococcus aureus.

A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3'-dimethoxybenzidine in the presence of copper. Disruption of mco showed copper sensitivity and H(2)O(2) resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.

Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain alpha-keto acid dehydrogenase.

Branched-chain fatty acids (BCFAs) typically constitute more than 90 % of the fatty acids of Listeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %) L. monocytogenes mutants (cld-1 and cld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917 was inserted into different genes of the branched-chain alpha-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, in which Tn917 was inserted into bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37 degrees C were stimulated by fatty acid precursors bypassing Bkd, 2-methylbutyrate (precursor for odd-numbered anteiso-fatty acids), isobutyrate (precursor for even-numbered iso-fatty acids) and isovalerate (precursor for odd-numbered iso-fatty acids). In contrast, the corresponding Bkd substrates, alpha-ketomethylvalerate, alpha-ketoisovalerate and alpha-ketoisocaproate, exhibited much poorer activity. At 26 degrees C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 degrees C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content of cld-2 from 33 % to 27 %, which may be close to the minimum BCFA requirement for L. monocytogenes. The transcription of bkd was enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors, cld-2 was able to increase its anteiso-C15 : 0 fatty acid content at 10 degrees C compared to 37 degrees C, which is the characteristic response of L. monocytogenes to low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis.

Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon.

The molecular events following inhibition of bacterial peptidoglycan synthesis have not been studied extensively. Previous proteomic studies have revealed that certain proteins are produced in increased amounts upon challenge of Staphylococcus aureus with cell-wall-active antibiotics. In an effort to further those studies, the genes upregulated in their expression in response to cell-wall-active antibiotics have been identified by genome-wide transcriptional profiling using custom-made Affymetrix S. aureus GeneChips. A large number of genes, including ones encoding proteins involved in cell-wall metabolism (including pbpB, murZ, fmt and vraS) and stress responses (including msrA, htrA, psrA and hslO), were upregulated by oxacillin, D-cycloserine or bacitracin. This response may represent the transcriptional signature of a cell-wall stimulon induced in response to cell-wall-active agents. The findings imply that treatment with cell-wall-active antibiotics results in damage to proteins including oxidative damage. Additional genes in a variety of functional categories were upregulated uniquely by each of the three cell-wall-active antibiotics studied. These changes in gene expression can be viewed as an attempt by the organism to defend itself against the antibacterial activities of the agents.

NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operon.

Staphylococcus aureus is a Gram-positive bacterium that is extremely halotolerant. To investigate the molecular mechanisms by which S. aureus can cope with osmotic stress, Tn917-lacZ-induced NaCl-sensitive mutants were isolated. An NaCl-sensitive mutant showed a longer lag period, slower growth rate, and lower final culture turbidity than the parent strain in liquid medium containing 1.5 M NaCl. Electron microscopic observation of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudo-multicellular cells. Addition of exogenous osmoprotectants, such as glycine betaine, choline, L-proline, and proline betaine, did not relieve the NaCl sensitivity of the mutant. The region flanking the transposon insertion site in the NaCl-sensitive S. aureus chromosome was sequenced. The mutated gene was 99% identical to arsR, the arsenic operon regulatory protein present on the pI258 plasmid of S. aureus. The ars operon from pI258 was subcloned into the shuttle vector pLI50 and transferred into the NaCl-sensitive mutant. The ars operon in trans restored NaCl tolerance in the mutant, suggesting that NaCl sensitivity is due to the mutation in arsR.

Impact of sigB mutation on Staphylococcus aureus oxacillin and vancomycin resistance varies with parental background and method of assessment.

Previous studies of Staphylococcus aureus transposon insertion mutants showing decreased methicillin or teicoplanin resistance have suggested a role for the RNA polymerase alternative sigma factor SigB in the expression of resistance to these antibiotics. A knockout mutation was created in the S. aureus strain COL sigB gene and its influence on oxacillin and vancomycin resistance was studied in a variety of parental backgrounds. Typically, sigB mutants of methicillin-resistant strains had oxacillin minimum inhibitory concentrations (MICs) one-half of their parent strains. The effect of the sigB mutation appeared to be more dramatic when assessed by population analysis profiles or by growth in liquid culture in shaking flasks than by MIC determinations. Oxacillin MICs of COL and the COLDeltasigB mutant were 400 and 200 mg/l, respectively, by conventional determination and 800 and 100-200 mg/l from population analysis profiles. The COLDeltasigB mutant strain was significantly more inhibited by a range of oxacillin concentrations in a shake flask culture than strain COL. Mutation of sigB caused a decrease in vancomycin resistance in two laboratory derived glycopeptide-intermediate S. aureus strains. The results suggest that some protein products whose expression is controlled by SigB play a role in resistance to cell wall-active antibiotics.

Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.

Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach.

Proteins produced in elevated amounts in response to oxacillin challenge of Staphylococcus aureus strain RN450, were studied by comparing Coomassie blue stained two-dimensional gels of cellular proteins. At least nine proteins were produced in elevated amounts following exposure to growth inhibitory concentrations of oxacillin. N-terminal sequences were obtained for five of the proteins and the databases were searched to tentatively identify them. The proteins were identified as homologs of (i) methionine sulfoxide reductase (MsrA); (ii) a signal transduction protein (TRAP) involved in regulating RNAIII production encoded by the agr locus; (iii) transcription elongation factor GreA; (iv) the heat shock protein GroES; and (v) the enzyme IIA component of the phosphoenolpyruvate:sugar phosphotransferase system. A similar induction response was observed with the other cell wall-active antibiotics, but not with antibiotics that affect other cellular targets. Increased transcription of the msrA and groEL genes in response to cell wall-active antibiotics was also demonstrated. Although net protein synthesis is inhibited subsequent to inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics, some proteins are induced in S. aureus, presumably in an attempt by the cell to counter the inhibitory effects of these agents.

Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus.

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.

Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds.

A series of 12 Staphylococcus aureus strains of various genetic backgrounds, methicillin resistance levels, and autolytic activities were subjected to selection for the glycopeptide-intermediate S. aureus (GISA) susceptibility phenotype on increasing concentrations of vancomycin. Six strains acquired the phenotype rapidly, two did so slowly, and four failed to do so. The vancomycin MICs for the GISA strains ranged from 4 to 16 microg/ml, were stable to 20 nonselective passages, and expressed resistance homogeneously. Neither ease of acquisition of the GISA phenotype nor the MIC attained correlated with methicillin resistance hetero- versus homogeneity or autolytic deficiency or sufficiency. Oxacillin MICs were generally unchanged between parent and GISA strains, although the mec members of both isogenic methicillin-susceptible and methicillin-resistant pairs acquired the GISA phenotype more rapidly and to higher MICs than did their susceptible counterparts. Transmission electron microscopy revealed that the GISA strains appeared normal in the absence of vancomycin but had thickened and diffuse cell walls when grown with vancomycin at one-half the MIC. Common features among GISAs were reduced doubling times, decreased lysostaphin susceptibilities, and reduced whole-cell and zymographic autolytic activities in the absence of vancomycin. This, with surface hydrophobicity differences, indicated that even in the absence of vancomycin the GISA cell walls differed from those of the parents. Autolytic activities were further reduced by the inclusion of vancomycin in whole-cell and zymographic studies. The six least vancomycin-susceptible GISA strains exhibited an increased capacity to remove vancomycin from the medium versus their parent lines. This study suggests that while some elements of the GISA phenotype are strain specific, many are common to the phenotype although their expression is influenced by genetic background. GISA strains with similar glycopeptide MICs may express individual components of the phenotype to different extents.

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Unique morphogenesis in Saccharomyces cerevisiae strain GS1731.

During the lag and early exponential phase of growth, 50-60% of budded cells of Saccharomyces cerevisiae strain GS1731 were multiply budded. During subsequent culture growth, the frequency of multiply budded cells decreased until by stationary phase multiply budded cells were rare. Data from renewed growth of a culture after hydroxyurea treatment indicated that GS1731 mother cells could assemble up to three pre-bud sites and begin bud growth and development in each. Light and scanning electron microscopy showed two or three very small buds emerging simultaneously on a mother cell and either reaching full size at the same time or enlarging sequentially. Immunofluorescence studies revealed that these multiply budded cells had multiple bundles of cytoplasmic microtubules. DAPI staining of nuclei revealed that some of the unbudded mother cells were multinucleate and completed cytokinesis giving rise to normal daughter cells.

Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus.

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of Staphylococcus aureus RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated zntR and zntA. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of zntA was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing zntR and zntA. An S. aureus strain harboring multiple copies of zntR and zntA showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that zntR and zntA genes were cotranscribed. The zinc uptake studies suggested that the zntA product was involved in the export of zinc ions out of cells.

Cloning and nucleotide sequencing of a Staphylococcus aureus gene encoding a branched-chain-amino-acid transporter.

We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889-1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureus chromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated alpha-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.

Molecular analysis of lytic genes of bacteriophage 80 alpha of Staphylococcus aureus.

Nucleotide sequencing of a 3779-bp fragment of the Staphylococcus aureus bacteriophage 80 alpha revealed two open reading frames: ORF1, designated as lytA, which encodes a polypeptide of 481 amino acids with an apparent M(r) of 53.81 kDa; and ORF2, designated as holin, which encodes for a hydrophobic polypeptide of 145 amino acids with an apparent M(r) of 15.58 kDa and exhibits two putative transmembrane helices. Both genes showed 100% sequence homology to that of the peptidoglycan hydrolase and holin genes of the S. aureus phage phi 11 reported earlier. In addition, the downstream sequences of the lytA gene were homologous to the phage attachment site (attP) of the phage phi 11. Based on our data we propose that the lytic system of the phage 80 alpha evolved from that of phage phi 11.

Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus.

A gene encoding an autolytic activity was identified in an autolysis-deficient mutant (Lyt-) of Staphylococcus aureus which produces only a single band in autolytic-activity gels (N. Mani, P. Tobin, and R. K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). An open reading frame, designated lytM, of 948 bp that could encode a polypeptide of 316 amino acid residues was identified. The calculated molecular mass of the lytM gene product (34.4 kDa) corresponded to that of the autolytic activity detected (approximately 36 kDa) in the Lyt- mutant. Results deduced from amino acid sequence analysis and N-terminal amino acid sequencing data suggest that LytM is a secreted protein. The C-terminal region of the putative protein encoded by lytM showed 51% identity with the N-terminal region of the mature lysostaphin from Staphylococcus simulans and 50% identity with the N-terminal region of ALE-1 from Staphylococcus capitis EPK1. Northern blot analysis showed that lytM expresses a transcript of approximately 955 bp, as predicted from the DNA sequence. Escherichia coli clones carrying the lytM gene exhibited autolytic-activity bands of approximately 36 kDa as well as of 19 and 22 kDa in activity gels. The lytM gene was mapped to the SmaI-D fragment on the S. aureus chromosome. Mapping data and results of hybridization experiments with primers generated from gene sequences of known autolytic genes of S. aureus clearly indicate that the lytM gene is distinct from other staphylococcal autolytic genes reported to date.