Predicting the minimum liquid surface tension activity of pseudomonads expressing biosurfactants.

UNLABELLED: Bacteria produce a variety of biosurfactants capable of significantly reducing liquid (aqueous) surface tension (γ) with a range of biological roles and biotechnological uses. To determine the lowest achievable surface tension (γMin ), we tested a diverse collection of Pseudomonas-like isolates from contaminated soil and activated sludge and identified those expressing biosurfactants by drop-collapse assay. Liquid surface tension-reducing ability was quantitatively determined by tensiometry, with 57 isolates found to significantly lower culture supernatant surface tensions to 24·5-49·1 mN m(-1) . Differences in biosurfactant behaviour determined by foaming, emulsion and oil-displacement assays were also observed amongst isolates producing surface tensions of 25-27 mN m(-1) , suggesting that a range of structurally diverse biosurfactants were being expressed. Individual distribution identification (IDI) analysis was used to identify the theoretical probability distribution that best fitted the surface tension data, which predicted a γMin of 24·24 mN m(-1) . This was in agreement with predictions based on earlier work of published mixed bacterial spp. data, suggesting a fundamental limit to the ability of bacterial biosurfactants to reduce surface tensions in aqueous systems. This implies a biological restriction on the synthesis and export of these agents or a physical-chemical restriction on their functioning once produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerous surveys of biosurfactant-producing bacteria have been conducted, but only recently has an attempt been made to predict the minimum liquid surface tension these surface-active agents can achieve. Here, we determine a theoretical minimum of 24 mN m(-1) by statistical analysis of tensiometry data, suggesting a fundamental limit for biosurfactant activity in bacterial cultures incubated under standard growth conditions. This raises a challenge to our understanding of biosurfactant expression, secretion and function, as well as being of interest to biotechnology where they are used in an increasingly wide range of applications.

Quorum-quenching activity of the AHL-lactonase from Bacillus licheniformis DAHB1 inhibits Vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality.

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.

Discontinuity of the incudo-stapedial joint within a fully aerated middle ear and mastoid on computed tomography: A clinico-radiological study of its aetiology and clinical consequence.

AIM: To investigate the aetiology and clinical consequences of incudo-stapedial (IS) discontinuity when it is demonstrated on computed tomography (CT) within a fully aerated middle ear and mastoid. METHODS AND MATERIALS: Patients with CT evidence of IS discontinuity within a fully aerated middle ear and mastoid were prospectively identified. Clinical history, otoscopic findings, audiometry, and CT data were evaluated. Predefined criteria were used to determine the likely aetiology of IS discontinuity, whether it was diagnosed prior to the CT study, and the clinical consequences in terms of degree of conductive hearing loss and requirement for surgical correction. The range of CT appearances was evaluated. RESULTS: The IS discontinuity in 34/36 ears was felt to be due to incus erosion secondary to chronic otitis, on the basis of clinical history and otoscopic findings. The IS discontinuity was rarely evident prior to CT with long-process deficiency being identified in only 5/36 cases. The mean air bone gap was only 22.5 dB. The ossicular defect was surgically addressed in only four cases. The incus deficiency was confined to the lower-third on CT in 19/36 cases. CONCLUSION: When IS discontinuity is demonstrated within a fully aerated middle ear and mastoid, the most likely aetiology is of acquired incus erosion due to chronic otitis media. The IS discontinuity on CT is usually not evident otoscopically. It usually results in only mild conductive hearing loss and the ossicular discontinuity was rarely surgically addressed in the present series.

Therapeutic potential of computer to cerebral cortex implantable devices.

In this article, an overview of some of the latest developments in the field of cerebral cortex to computer interfacing (CCCI) is given. This is posed in the more general context of Brain-Computer Interfaces in order to assess advantages and disadvantages. The emphasis is clearly placed on practical studies that have been undertaken and reported on, as opposed to those speculated, simulated or proposed as future projects. Related areas are discussed briefly only in the context of their contribution to the studies being undertaken. The area of focus is notably the use of invasive implant technology, where a connection is made directly with the cerebral cortex and/or nervous system. Tests and experimentation which do not involve human subjects are invariably carried out a priori to indicate the eventual possibilities before human subjects are themselves involved. Some of the more pertinent animal studies from this area are discussed. The paper goes on to describe human experimentation, in which neural implants have linked the human nervous system bidirectionally with technology and the internet. A view is taken as to the prospects for the future for CCCI, in terms of its broad therapeutic role.

The structure-function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain.

The GGDEF response regulator WspR couples the chemosensory Wsp pathway to the overproduction of acetylated cellulose and cell attachment in the Pseudomonas fluorescens SBW25 wrinkly spreader (WS) genotype. Here, it is shown that WspR is a diguanylate cyclase (DGC), and that DGC activity is elevated in the WS genotype compared to that in the ancestral smooth (SM) genotype. A structure-function analysis of 120 wspR mutant alleles was employed to gain insight into the regulation and activity of WspR. Firstly, 44 random and defined pentapeptide insertions were produced in WspR, and the effects determined using assays based on colony morphology, attachment to surfaces and cellulose production. The effects of mutations within WspR were interpreted using a homology model, based on the crystal structure of Caulobacter crescentus PleD. Mutational analyses indicated that WspR activation occurs as a result of disruption of the interdomain interface, leading to the release of effector-domain repression by the N-terminal receiver domain. Quantification of attachment and cellulose production raised significant questions concerning the mechanisms of WspR function. The conserved RYGGEEF motif of WspR was also subjected to mutational analysis, and 76 single amino acid residue substitutions were tested for their effects on WspR function. The RYGGEEF motif of WspR is functionally conserved, with almost every mutation abolishing function.

C-terminal interactions between the XerC and XerD site-specific recombinases.

Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed. Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold. Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.

Relating primary structure to function in the Escherichia coli XerD site-specific recombinase.

XerC and XerD are related 298-amino-acid site-specific recombinases, each of which is responsible for the exchange of one pair of strands in Xer recombination. Both recombinases encode functions necessary for sequence-specific DNA-binding, co-operative XerC/D interactions, synapsis and catalysis. These functions were related to the primary amino acid sequence by constructing and analysing internal and C-terminal XerD deletions. An XerD derivative containing residues 1-233 was proficient in specific DNA binding, but did not interact co-operatively with XerC. Deletion of a further five C-terminal amino acids abolished binding to DNA. Proteins deleted for residues 32-88 and for residues 145-159 were deficient in DNA binding. Deletion of residues 244-281, a region containing amino acids necessary for catalysis, gave a protein that bound to DNA. An XerD derivative containing residues 1-268 retained co-operative interactions with XerC; nevertheless, it did not support XerC strand exchange and was defective in XerD catalysis. Residues 1-283 retain a functional catalytic active site, though a protein lacking the five C-terminal amino acids was still unable to mediate normal in vivo recombination, indicating that these residues are needed for a function that is not directly related to DNA binding or catalysis.

Regulatory interactions between RepA, an essential replication protein, and the DNA repeats of RepFIB from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene (BCDD'D" and EFGHIJ). In vivo genetic tests demonstrate that the BCDD'D" repeats form part of the origin of replication, while some of the downstream repeat elements (HIJ) are involved in the sensing and setting of plasmid copy number. RepA DNA binding to these groups of repeats has been investigated in vivo by utilizing the fact that the replicon contains three active promoters (orip, repAp, and EFp), one of which has previously been shown to control the expression of repA (repAp). All three promoters are closely associated with the repeat elements flanking repA, and an investigation using lacZ or cml gene fusions has demonstrated that RepA expressed in trans is able to repress each promoter. However, these assays suggest that the transcriptional responses of orip and repAp to RepA repression are significantly different, despite the fact that both promoters are embedded within the BCDD'D" repeat elements. Extra copies of the BCDD'D" or EFG repeats in trans have no effect on RepA repression of repAp embedded in a second copy of the BCDD'D" repeats, but copies of the HIJ or EFGHIJ repeats are able to derepress repAp, suggesting that there is a fundamental difference between RepA-BCDD'D" or -HIJ complexes and RepA-EFG or -EFGHIJ complexes.

RepFIB: a basic replicon of large plasmids.

The distribution of the RepFIB replicon among a total of 20 plasmid incompatibility groups was determined using Southern blot and polymerase chain reaction analysis. The presence of the replicon was confirmed in 25 of 55 plasmids tested. The majority of plasmids carrying RepFIB are from the IncF incompatibility groups, but one plasmid from IncI and one plasmid from IncP also possess the replicon. Seven different examples of RepFIB were sequenced in the minimal replicon region to obtain 1525 bp of sequence information covering the repA gene and flanking repeat regions for comparison. An analysis of these sequences plus three sequences previously reported showed almost perfect conservation of the predicted protein sequence of RepA and of the flanking DNA repeats. DNA sequence data were analyzed using maximum parsimony techniques to describe the possible evolutionary relationships of the 10 examples of RepFIB.

Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene. Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream. Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter. The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids. Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence.

Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids.

A second autonomous replicon of P307, RepFIB, has been isolated that has significant homology with other replicons in IncFI group plasmids. Eleven homologous repeats of 21 base pairs are present on the sequence and flank an open reading frame capable of coding for a protein of about Mr = 40,000. This protein was identified by maxicell analysis of cloned RepFIB. A series of deletion mutations of RepFIB were inserted into a DNA polymerase I-dependent vector and examined for their replication proficiency in a polA1 strain. These experiments defined a minimal replication region of 1.6 kilobases which includes the three repeats immediately upstream and downstream of the open reading frame. Deletion of a second set of repeats further downstream doubled the copy number of a chimeric plasmid replicating under RepFIB control. It was concluded that these repeats control the copy number of the replicon. Incompatibility tests showed that all three sets of repeats could express incompatibility with a resident RepFIB plasmid.