Roger Simm

Phenotypic convergence mediated by GGDEF-domain-containing proteins.

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.

A Role for the EAL-Like Protein STM1344 in Regulation of CsgD Expression and Motility in Salmonella enterica Serovar Typhimurium

The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.

Regulation of biofilm formation in Salmonella enterica serovar Typhimurium

In animals, plants and the environment, Salmonella enterica serovar Typhimurium forms the red dry and rough (rdar) biofilm characterized by extracellular matrix components curli and cellulose. With complex expression control by at least ten transcription factors, the bistably expressed orphan response regulator CsgD directs rdar morphotype development. CsgD expression is an integral part of the Hfq regulon and the complex cyclic diguanosine monophosphate signaling network partially controlled by the global RNA-binding protein CsrA. Cell wall turnover and the periplasmic redox status regulate csgD expression on a post-transcriptional level by unknown mechanisms. Furthermore, phosphorylation of CsgD is a potential inactivation and degradation signal in biofilm dissolution. Including complex incoherent feed-forward loops, regulation of biofilm formation versus motility and virulence is of recognized complexity.

BC4707 Is a Major Facilitator Superfamily Multidrug Resistance Transport Protein from Bacillus cereus Implicated in Fluoroquinolone Tolerance

Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.

Dissecting the cyclic di-guanylate monophosphate signalling network regulating motility inSalmonella entericaserovar Typhimurium: c-di-GMP signalling regulating motility inS. sv. Typhimurium

Flagella-mediated swimming and swarming motility in Salmonella enterica serovar Typhimurium is intercalated with the cyclic di-guanylate monophosphate (c-di-GMP) signalling network. In this study, we identified the GGDEF domain proteins STM2672, STM4551 and STM1987 as key di-guanylate cyclases involved in regulation of motility in a ΔyhjH phosphodiesterase gene deletion mutant with elevated c-di-GMP levels inhibiting motility. Surprisingly, these di-guanylate cyclases distinctively inhibited motility through the c-di-GMP receptors YcgR and the cellulose synthase BcsA, whereby STM2672 corresponded to YcgR, STM1987 to BcsA and STM4551 to both receptors. Although downregulation of motility is believed to prepare the bacterial cells for surface adhesion and biofilm formation, the major biofilm regulator CsgD of S. sv. Typhimurium was not involved in the regulation of swimming or swarming motility. Together with previously identified c-di-GMP networks regulating flagella-related phenotypes, flagella biosynthesis is a major target of c-di-GMP signalling in S. sv. Typhimurium.

SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins

The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

The ERM Proteins Ezrin and Moesin Regulate Retrograde Shiga Toxin Transport

The ERM proteins (ezrin, radixin and moesin) are known for connecting the actin cytoskeleton to the plasma membrane. They have been found to associate with lipid rafts as well as to be important for endosomal sorting and receptor signaling. However, little is known about the role of ERM proteins in retrograde transport and lipid homeostasis. In this study, we show that ezrin and moesin are important for efficient cell surface association of Shiga toxin (Stx) as well as for its retrograde transport. Furthermore, we show that depletion of these proteins influences endosomal dynamics and seems to enhance Stx transport toward lysosomes. We also show that knockdown of Vps11, a subunit of the HOPS complex, leads to increased retrograde Stx transport and reverses the inhibiting effect of ezrin and moesin knockdown. Importantly, retrograde transport of the plant toxin ricin, which binds to both glycolipids and glycoproteins with a terminal galactose, seems to be unaffected by ezrin and moesin depletion.

Gene transcription from the linear plasmid pBClin15 leads to cell lysis and extracellular DNA-dependent aggregation of Bacillus cereus ATCC 14579 in response to quinolone-induced stress.

The Bacillus cereus type strain ATCC 14579 harbours pBClin15, a linear plasmid with similar genome organization to tectiviruses. Since phage morphogenesis is not known to occur it has been suggested that pBClin15 may be a defect relic of a tectiviral prophage without relevance for the bacterial physiology. However, in this paper, we demonstrate that a pBClin15-cured strain is more tolerant to antibiotics interfering with DNA integrity than the WT strain. Growth in the presence of crystal violet or the quinolones nalidixic acid, norfloxacin or ciprofloxacin resulted in aggregation and lysis of the WT strain, whereas the pBClin15-cured strain was unaffected. Microarray analysis comparing the gene expression in the WT and pBClin15-cured strains showed that pBClin15 gene expression was strongly upregulated in response to norfloxacin stress, and coincided with lysis and aggregation of the WT strain. The aggregating bacteria experienced a significant survival benefit compared with the planktonic counterparts in the presence of norfloxacin. There was no difference between the WT and pBClin15-cured strains during growth in the absence of norfloxacin, the pBClin15 genes were moderately expressed, and no effect was observed on chromosomal gene expression. These data demonstrate for the first time that although pBClin15 may be a remnant of a temperate phage, it negatively affects the DNA stress tolerance of B. cereus ATCC 14579. Furthermore, our results warrant a recommendation to always verify the presence of pBClin15 following genetic manipulation of B. cereus ATCC 14579.

Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids.

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.

A new family of proteins related to the HEAT-like repeat DNA glycosylases with affinity for branched DNA structures

The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique β-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.