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.

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.

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.

Sequence analysis of the region downstream from a peptidoglycan hydrolase-encoding gene from Staphylococcus aureus NCTC8325.

The nucleotide (nt) sequence of a 4.7-kb DNA fragment downstream from a peptidoglycan hydrolase-encoding gene (lytA) from Staphylococcus aureus NCTC8325 was determined. Sequencing revealed three open reading frames (ORFs) of 513, 447 and 879 bp with consensus ribosome-binding sites located upstream from the ATG start codons. Results from in vitro transcription-translation analysis and maxicell experiments suggested that the 447-bp ORF was the one being actively expressed. Comparison of the amino acid (aa) sequences of the ORFs with the aa sequences in the NCBI Entrez database (Release 4.0, April 1993) did not show any significant homology to any sequenced polypeptides. However, nt sequences downstream from lytA showed perfect homology to the bacteriophage phi 11 attachment site (attP) and integration site (attB), and significant homology to downstream regions of the staphylokinase (sak) and exfoliative toxin A (eta) genes of S. aureus.

Sequence analysis of the region upstream of a peptidoglycan hydrolase-encoding gene from bacteriophage phi 11 of Staphylococcus aureus.

The nucleotide sequence of a 1.1-kb DNA fragment upstream of a peptidoglycan hydrolase-encoding gene (lytA) from bacteriophage phi 11 of Staphylococcus aureus was determined to see if the upstream sequences are involved in the transfer of the lytA product through the cytoplasmic membrane. Sequencing revealed three open reading frames of 171, 147 and 435 bp with consensus Shine-Dalgarno sequences located upstream from the ATG start codons. The third open reading frame overlaps with the 5' end of lytA by 18 nucleotides. Comparison of the deduced amino acid sequences of the open reading frames with the amino acid sequences in the NCBI Entrez database did not show any significant homology to any sequenced polypeptides. However, the analysis of the peptides showed some structural similarities to the product of the holin gene family. Lysogens containing an insertional mutation in ORF3, upon induction, produced either no phage titer or very low phage titers, compared to the wild-type lysogen. Transformation of ORF3 mutated lysogens by a plasmid containing the intact ORF3 produced the same phage titer as wild-type lysogen, suggesting that the ORF3 product is involved in the process of cell lysis/phage release.

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.

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.

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.

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 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.

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.

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.

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.

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.

Cell-density-dependent Changes in the Metabolism of Chloronema Cell Cultures: I. Relationship between Cell Density and Enzymic Activities.

In the growing chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw., activities of several enzymes have been found to be cell-density-dependent. Cyclic nucleotide phosphodiesterase (cNPDE), nitrate reductase (NR), and protein kinase showed highest activity at a low cell density (1 to 2 milligrams per milliliter) while indoleacetic acid (IAA) oxidase and peroxidase were highest at a high cell density (>10 milligrams per milliliter). 3'-Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.In moss protonema, the dependence of cNPDE, IAA oxidase, and peroxidase on cell density may play an important role in modulating the endogenous levels of IAA and cAMP, both of which regulate the differentiation of specific cell types (Johri and Desai 1973 Nature New Biol 245: 223-224; and Handa and Johri 1976 Nature 259: 480-482).

Environmental factors affecting the antagonism of Pseudomonas cepacia against Trichoderma viride.

Antagonistic activity of the bacterium Pseudomonas cepacia against Trichoderma viride was greatly influenced by nutritional and environmental conditions. Xylose and trehalose strongly enhanced the antifungal activity of P. cepacia, whereas mannitol and glucose had little effect. The carbon sources that enhanced the antagonistic activity also inhibited sporulation of T. viride. Antagonism of P. cepacia was enhanced by ammonium nitrogen; however, with nitrite or nitrate there was only a little antagonism. The antagonism of P. cepacia was optimal at pH 5.0. Although P. cepacia showed maximum antagonism against T. viride at 37 degrees C, the antagonism was fairly good at temperatures as low as 18 degrees C, indicating that there is a broad range of temperature for the antifungal activity of P. cepacia.

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.

Isolation and characterization of a pseudomonas strain that restricts growth of various phytopathogenic fungi.

The characterization of a novel Pseudomonas strain exhibiting antagonism towards many important corn fungal pathogens is presented. This strain was isolated from the caryopses of the grass Tripsacum dactyloides and was identified as Pseudomonas cepacia. The antagonistic activity is due to the production of an antifungal compound. The chromatographic properties of this partially purified compound isolated from growth medium differ from those reported previously for other pseudomonads. The suppression of the growth of economically important phytopathogens by this strain and by the partially purified compound indicates a potential biocontrol agent.