Bacterial metabolic 'toxins': a new mechanism for lactose and food intolerance, and irritable bowel syndrome.

Lactose and food intolerance cause a wide range of gut and systemic symptoms, including gas, gut pain, diarrhoea or constipation, severe headaches, severe fatigue, loss of cognitive functions such as concentration, memory and reasoning, muscle and joint pain, heart palpitations, and a variety of allergies (Matthews and Campbell, 2000; Matthews et al., 2005; Waud et al., 2008). These can be explained by the production of toxic metabolites from gut bacteria, as a result of anaerobic digestion of carbohydrates and other foods, not absorbed in the small intestine. These metabolites include alcohols, diols such as butan 2,3 diol, ketones, acids, and aldehydes such as methylglyoxal (Campbell et al., 2005, 2009). These 'toxins' induce calcium signals in bacteria and affect their growth, thereby acting to modify the balance of microflora in the gut (Campbell et al., 2004, 2007a,b). These bacterial 'toxins' also affect signalling mechanisms in cells around the body, thereby explaining the wide range of symptoms in people with food intolerance. This new mechanism also explains the most common referral to gastroenterologists, irritable bowel syndrome (IBS), and the illness that afflicted Charles Darwin for 50 years (Campbell and Matthews, 2005a,b). We propose it will lead to a new understanding of the molecular mechanism of type 2 diabetes and some cancers.

Importance of eps genes from Bacillus subtilis in biofilm formation and swarming.

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming in Bacillus subtilis strain 168. To assess the role of particular proteins encoded by the group of epsI-epsO genes that form the eps operon, we constructed a series of insertional mutants. The results obtained showed that mutations in epsJ-epsN, but not in the last gene of the eps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of the sfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), the sfp+ strains defective in eps genes (except epsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168 sfp+.

Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli.

Verapamil is used clinically as a Ca(2+) channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.

Molecular insights into the mechanism of ATP-hydrolysis by the NBD of the ABC-transporter HlyB.

The ABC-transporter HlyB is a central element of the Type I protein secretion machinery, dedicated to export the E. coli toxin HlyA in a single step across the two membranes of the cell envelope. Here, we discuss recent insights into the structure and the mechanism of ATP-hydrolysis by the NBD of HlyB. Combining structural and biochemical data, we have suggested that substrate-assisted catalysis (SAC), but not general base catalysis, is responsible for ATP-hydrolysis in this NBD and might also operate in other NBDs. Finally, the implications and advantages of SAC are discussed in the context of ATP-induced dimerization of the NBDs.

Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin.

HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.

A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB.

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae: Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis. mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquid cultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection. Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA. These results indicate the complex regulation of the mrf cluster during infection.

The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain.

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance.

Knockout mutations were constructed in the arcA gene of a virulent type b strain of Haemophilus influenzae, and the behavior of the resulting mutants was investigated in a number of conditions that mimicked distinct steps in the natural infection pathway. In arcA mutants, synthesis of capsule and lipooligosaccharide (LOS) and growth in synthetic media were unaltered compared to synthesis of capsule and LOS and growth in synthetic media in the wild-type H. influenzae type b parent strain. However, the virulence of the arcA mutants for BALB/c mice was significantly reduced. Upon exposure to human blood or serum, the arcA mutants showed markedly reduced survival compared with the survival of its wild-type parent. Serum resistance could be fully restored by complementation in cis with the H. influenzae arcA gene but not by complementation in cis with the homologous gene from Escherichia coli. The proteomes of wild-type and mutant bacteria were markedly different, especially under anaerobic conditions, underscoring the global regulatory role of ArcAB in H. influenzae. Evaluation of antibody titers and classical complement activities in various serum samples pointed to complement-mediated bactericidal activity as the factor that distinguishes between the arcA mutant and wild-type phenotypes. Comparative analysis of the membrane fractions of the arcA mutants and the wild-type strain revealed several ArcA-regulated proteins, some of which may be implicated in the serum hypersensitivity phenotype.

Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli.

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12.

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

An assessment of the role of intracellular free Ca2+ in E. coli.

We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters. In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca(2+)-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca(2+). However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca(2+). Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx. The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.

ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans.

The approximately 27 kDa ABC-ATPase, an extraordinarily conserved, unique type of ATPase, acts as a machine to fuel the movement across membranes of almost any type of molecule, from large polypeptides to small ions, via many different membrane-spanning proteins. A particular ABC-ATPase must therefore be tailor-made to function in a complex with its cognate membrane protein, forming a transport pathway appropriate for a specific type of molecule, or in the case of some ABC-transporters, several types of molecule. Molecules to be transported recognise their own transporter, bind and switch on the ATPase, which in turn activates or opens the transport pathway. ABC-dependent transport can be inwards across the membrane, or outwards to the cell exterior, and the ABC-ATPase can fuel transport through pathways which may involve a classical channel (CFTR), a "gateway" mechanism through a proteinacious chamber spanning the bilayer, or conceivably via a pathway at the protein-lipid interface of the outside of the membrane domain. This may be the case for drugs transported by Pgp, a multidrug resistance transporter. In this review, we try to identify the common fundamental principles which unite all ABC-transporters, including the basis of specificity for different transported compounds (allocrites), the interactions between the ATPase and membrane domains, activation of the ATPase and the coupling of consequent conformational changes, to the final movement of an allocrite through a given transport pathway. We discuss the so far limited structural information for the intact ABC-transporter complex and the exciting information from the first crystal structure of an ABC-ATPase. Finally, the action of specific transporters, CFTR (Cl- transport), Pgp, MRP and LmrA, all transporting many different drug molecules and HlyB transporting a large protein toxin are discussed.

Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.

Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.

Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator.

HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC. HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37 degrees C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites. However, the effect of removing both HlyB and TolC is not additive. On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC. This suggests that the presence of HlyB induces a structural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does. This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective. Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active haemolysin is limited to a brief period during mid to late exponential phase. In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion.

Increased sensitivity of E. coli to novobiocin, EDTA and the anticalmodulin drug W7 following overproduction of DjlA requires a functional transmembrane domain.

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required. the full-length, wild-type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway.

DjIA is a novel DnaJ-like protein localized to the inner membrane of Escherichia coli through the single transmembrane domain (TMD) found at the N-terminus. The overproduction of DjIA activates expression of the cps operon, controlling synthesis and export of the extracellular polysaccharide colanic acid via the Rcs/B two-component signal transduction pathway. We now show that both the TMD and the J-region are essential for the induction of cps expression observed with the overproduction of DjIA. Furthermore, we describe the isolation and characterization of different point mutations in the TMD that completely or partially block the induction of cps expression associated with overproduction of DjIA. These mutations were shown not to affect the localization, stability or topology of the mutant DjIA proteins. We propose that these mutations are affecting specific interactions between the TMD of DjIA and its substrate protein(s), for example RcsC, the membrane sensor kinase partner of the Rcs/B signal transduction pathway.

Artefactual cleavage of E coli H-NS by OmpT.

In the bacterium Escherichia coli, H-NS-(H1, H1a) is a heat-stable protein with a molecular mass of 15.5 kDa involved in nucleoid organisation and gene regulation linked to certain signal transduction pathways. We have shown that, following addition of preparations of everted inner membrane vesicles, heat-stable cleavage products of approximately 10 kDa of H-NS are formed in vitro from newly synthesised, radio-labelled H-NS and from purified H-NS. The 15.5 kDa protein and its cleavage products were also recovered from a minicell system. These results raised the possibility that cleavage of H-NS is physiologically significant. However, the cleavage of H-NS observed appears to occur during cell breakage and to depend on the method of protein extraction and the presence of the outer membrane protease, OmpT. Nevertheless, the results indicate that H-NS may contain at least two separate domains with cleavage occurring between these domains at a preferred OmpT site. Failure to take account of H-NS cleavage in sample preparation and analysis can lead to serious underestimation of H-NS levels.