William L. Kelley

The J-domain family and the recruitment of chaperone power

The defining feature of the Hsp40 chaperone family is a approximately 70-amino-acid-residue signature, termed the J domain, that is necessary for orchestrating interactions with its Hsp70 chaperone partner(s). J-domain proteins play important regulatory roles as co-chaperones, recruiting Hsp70 partners and accelerating the ATP-hydrolysis step of the chaperone cycle. Certain proteins could have acquired a J domain in order to present a specific substrate(s) to an Hsp70 partner and thus capitalize upon chaperone activities when carrying out cellular functions. J-domain proteins participate in complex biological processes, such as cell-cycle control by DNA tumor viruses, regulation of protein kinases and exocytosis.

Staphylococcus aureus: new evidence for intracellular persistence

Many reports have documented that Staphylococcus aureus can invade host cells and persist intracellularly for various periods of time in cell culture models. However, it is not clear whether intracellular persistence of S. aureus also occurs in the course of infections in whole organisms. This is a subject of intense debate and is difficult to assess experimentally. Intracellular persistence would provide S. aureus with an ideal strategy to escape from professional phagocytes and extracellular antibiotics and would promote recrudescent infection. Here, we present a brief overview of the mounting evidence that S. aureus has the potential to internalize and survive within host cells.

The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions

Molecular chaperones are highly conserved in all free‐living organisms. There are many types of chaperones, and most are conveniently grouped into families. Genome sequencing has revealed that many organisms contain multiple members of both the DnaK (Hsp70) family and their partner J‐domain protein (JDP) cochaperone, belonging to the DnaJ (Hsp40) family. Escherichia coli K‐12 encodes three Hsp70 genes and six JDP genes. The coexistence of these chaperones in the same cytosol suggests that certain chaperone–cochaperone interactions are permitted, and that chaperone tasks and their regulation have become specialized over the course of evolution. Extensive genetic and biochemical analyses have greatly expanded knowledge of chaperone tasking in this organism. In particular, recent advances in structure determination have led to significant insights of the underlying complexities and functional elegance of the Hsp70 chaperone machine.

Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by hemB Mutants of Staphylococcus aureus Expressing Small Colony Variant Phenotypes

ABSTRACT Small colony variants (SCVs) of Staphylococcus aureus are slow-growing subpopulations that cause persistent and relapsing infections. The altered phenotype of SCV can arise from defects in menadione or hemin biosynthesis, which disrupt the electron transport chain and decrease ATP concentrations. With SCVs, virulence is altered by a decrease in exotoxin production and susceptibility to various antibiotics, allowing their intracellular survival. The expression of bacterial adhesins by SCVs is poorly documented. We tested fibrinogen- and fibronectin-mediated adhesion of a hemB mutant of S. aureus 8325-4 that is defective for hemin biosynthesis and exhibits a complete SCV phenotype. In this strain, adhesion to fibrinogen and fibronectin was significantly higher than that of its isogenic, normally growing parent and correlated with the increased surface display of these adhesins as assessed by flow cytometry. Real-time quantitative reverse transcription-PCR demonstrated increased expression of clfA and fnb genes by the hemB mutant compared to its isogenic parent. The influence of the hemB mutation on altered adhesin expression was confirmed by showing complete restoration of the wild-type adhesive phenotype in the hemB mutant, either by complementing with intact hemB or by supplementing the growth medium with hemin. Increased surface display of fibrinogen and fibronectin adhesins by the hemB mutation occurred independently from agr, a major regulatory locus of virulence factors in S. aureus. Both agr-positive and agr-lacking hemB mutants were also more efficiently internalized by human embryonic kidney cells than were their isogenic controls, presumably because of increased surface display of their fibronectin adhesins.

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BackgroundStaphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown.ResultsWe have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited.ConclusionThese molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon

The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome‐wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA‐like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage‐inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon‐controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by β‐lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two‐component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic‐induced SOS response and its consequences.

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

ABSTRACT Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-β lactamase/β-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

Recognition, Targeting, and Hydrolysis of the λ O Replication Protein by the ClpP/ClpX Protease

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage λ O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of λ O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of λ O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with λ O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis

The contribution of in vivo biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis was studied in an experimental model of foreign body infections. Increasing inocula (from 10(2) to 10(7) organisms) of ica-positive strains of S. aureus and S. epidermidis and their ica-negative isogenic mutants (the ica locus codes for a major polysaccharide component of biofilm) were injected into subcutaneously implanted tissue cages in guinea pigs. Surprisingly, bacterial counts and time-course of tissue cage infection by ica-positive strains of S. aureus or S. epidermidis were equivalent to those of their respective ica-negative mutants, in the locally infected fluids and on tissue-cage-inserted plastic coverslips.

Coordinated Regulation by AgrA, SarA, and SarR To Control agr Expression in Staphylococcus aureus

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that AgrA alone promoted transcription from the agr P2 promoter, with SarA enhancing it and SarR inhibiting agr P2 transcription in the presence of AgrA or with SarA and AgrA. Electrophoretic mobility shift assay (EMSA) analysis disclosed that SarR binds more avidly to the agr promoter than SarA and displaces SarA from the agr promoter. Additionally, SarA and AgrA bend the agr P2 promoter, whereas SarR does not. Collectively, these data indicated that AgrA activates agr P2 and P3 promoters while SarA activates the P2 promoter, presumably via bending of promoter DNA to bring together AgrA dimers to facilitate engagement of RNA polymerase (RNAP) to initiate transcription.