Hanne Ingmer

H‐NS: a modulator of environmentally regulated gene expression

H‐NS is a small chromatin‐associated protein found in enterobacteria. H‐NS has affinity for all types of nucleic acids but binds preferentially to intrinsically curved DNA. The major role of H‐NS is to modulate the expression of a large number of genes, mostly by negatively affecting transcription. Many of the H‐NS‐modulated genes are regulated by environmental signals, and expression of most of these genes is positively regulated by specific transcription factors. Therefore one of the purposes of H‐NS could be to repress expression of some genes under conditions characteristic of a non‐intestinal environment, but allow expression of specific genes in response to certain stimuli in the intestinal environment. The hns gene is autoregulated. In vivo the H‐NS to DNA ratio is fairly constant except during cold shock, when it increases three‐ to fourfold. In this review we propose that only the preferential binding to intrinsically curved DNA plays a role under normal growth conditions, and we discuss the different mechanisms by which H‐NS might affect gene expression and how H‐NS could be involved in the response to different stress situations. Finally, we summarize the evolutionary and functional relationship between H‐NS and the homologous StpA.

Proteolytic systems of lactic acid bacteria.

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.

The RNA-Binding Protein Hfq of Listeria monocytogenes: Role in Stress Tolerance and Virulence

In gram-negative bacteria, the RNA-binding protein Hfq has emerged as an important regulatory factor in a variety of physiological processes, including stress resistance and virulence. In Escherichia coli, Hfq modulates the stability or the translation of mRNAs and interacts with numerous small regulatory RNAs. Here, we studied the role of Hfq in the stress tolerance and virulence of the gram-positive food-borne human pathogen Listeria monocytogenes. We present evidence that Hfq is involved in the ability of L. monocytogenes to tolerate osmotic and ethanol stress and contributes to long-term survival under amino acid-limiting conditions. However, Hfq is not required for resistance to acid and oxidative stress. Transcription of hfq is induced under various stress conditions, including osmotic and ethanol stress and at the entry into the stationary growth phase, thus supporting the view that Hfq is important for the growth and survival of L. monocytogenes in harsh environments. The stress-inducible transcription of hfq depends on the alternative sigma factor sigmaB, which controls the expression of numerous stress- and virulence-associated genes in L. monocytogenes. Infection studies showed that Hfq contributes to pathogenesis in mice, yet plays no role in the infection of cultured cell lines. This study provides, for the first time, information on the role of Hfq in the stress tolerance and virulence of a gram-positive pathogen.

Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus

The Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus. Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus, but not for survival during heat shock. In the present study, we have inactivated clpC, clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heat‐shock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.

Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria.

Clp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, mutants in the clp genes in the low‐GC‐content Gram‐positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpP‐containing proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non‐stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), σH and Sda (sporulation). Thus, Clp proteins are central in co‐ordinating developmental decisions and stress response in low GC Gram‐positive bacteria.

Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence

Clp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature‐tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325‐4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall‐associated proteins. In the mutant cells, the amount of α‐haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum‐sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.

ClpP participates in the degradation of misfolded protein in Lactococcus lactis

ClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms. In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases. We show here that the clpP gene from the Gram‐positive bacterium Lactococcus lactis encodes a 22‐kDa protein that is induced by low pH and by the t‐RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl‐containing peptides. Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram‐positive bacteria. When we disrupted the L. lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild‐type cells. Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl‐containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress. Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.

Global Virulence Regulation in Staphylococcus aureus: Pinpointing the Roles of ClpP and ClpX in the sar/agr Regulatory Network

ABSTRACT Staphylococcus aureus causes infections ranging from superficial wound infections to life-threatening systemic infections. Essential for S. aureus pathogenicity are a number of cell-wall-associated and secreted proteins that are controlled by a complex regulatory network involving the quorum-sensing agr locus and a large set of transcription factors belonging to the Sar family. Recently, we revealed a new layer of regulation by showing that mutants lacking the ClpXP protease produce reduced amounts of several extracellular virulence factors and that, independently of ClpP, ClpX is required for transcription of spa, encoding Protein A. Here we find that the independent effect of ClpX is not general for other cell wall proteins, as expression of fibronectin- and fibrinogen-binding proteins was increased in the absence of either ClpX or ClpP. To assess the roles of ClpX and ClpP within the sar/agr regulatory network, deletions in clpX and clpP were combined with mutations in these genes. Interestingly, the derepression of spa transcription normally observed in an agr-negative strain was abolished in cells devoid of ClpX, and apparently ClpX modulates both SarS-dependent and SarS-independent control of spa expression, perhaps through the Sar family member Rot. Examination of expression of a single secreted protein, the SspA serine protease, revealed that ClpXP, similar to agr, is required for growth phase-dependent transcriptional induction of sspa. Intriguingly, induction was restored by the concomitant inactivation of Rot. We hypothesize that RNAIII accumulating in the postexponential phase may target Rot for degradation by ClpXP, leading to derepression of sspA.

Identification of proteins induced at low pH in Lactococcus lactis.

The Gram-positive bacterium Lactococcus lactis is of major importance to the dairy industry due to its conversion of lactose to lactic acid leading to the acidification of milk. To investigate which proteins are induced when L. lactis is exposed to conditions of low pH, we used two-dimensional gel electrophoresis to follow how protein expression changes with the degree of acidification. We found that reducing the pH of the growth medium with hydrochloric acid induced the synthesis of a small subset of proteins. The majority of these proteins were induced both after a minor (pH 5.5) and a major (pH 4.5) reduction in pH. Among the most strongly induced proteins, we identified the oxidative stress proteins superoxide dismutase and alkylhydroperoxidase as well as the autoinducer synthesis protein, LuxS. We also observed a differential induction of heat shock proteins by low pH as members of the CtsR regulon, ClpE and ClpP were induced at both pH 5.5 and 4.5, while HrcA-regulated chaperones, GroEL, GroES, DnaK and GrpE were induced only at pH 4.5. In addition, we identified two proteins repressed by low pH that proved to be the L. lactis HPr protein of the phosphoenolpyruvate sugar phosphotransferase system and the trigger factor known to participate in the folding of newly synthesized polypeptides.

BACTERIAL CHITINASES AND CHITIN-BINDING PROTEINS AS VIRULENCE FACTORS

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.