Feasibility of biventricular 3D transthoracic echocardiography in the critically ill and comparison with conventional parameters.

BACKGROUND: Transthoracic 3D cardiac analysis is enticing in its potential simplicity and wealth of data available. It has been suggested to be accurate vs magnetic resonance imaging in relatively stable patients, but feasibility and agreement with conventional echocardiographic assessment of stroke volume (SV) have not been thoroughly assessed in critically ill patients, who are traditionally harder to image. The objectives of this study were to compare 3D transthoracic volumetric analysis vs Doppler assessment of SV (which is suggested to be accurate in the critically ill) and Simpson's biplane assessment in a cohort typical of the intensive care unit (ICU), where accurate assessment is important: mechanically ventilated patients with a significant ventilation/perfusion (V/Q) mismatch. We hypothesised that it would be feasible but might lack agreement. METHODS: Patients were imaged within 24 hours of admission. Inclusion criteria were adult patients, V/Q mismatch present (defined as a ratio of arterial oxygen partial pressure to fractional inspired oxygen < 300), and mechanically ventilated with Doppler SV assessment possible. Biventricular echocardiographic volumetric analysis was performed using Siemens SC2000 along with standard Simpson's biplane and Doppler SV assessment. 3D images were unacceptable if two segments or more were unable to be seen in two volumetric planes. 3D left ventricular (3DLV) and 3D right ventricular (3DRV) analyses were performed with the Tomtec Imaging and Siemens Acuson platforms, respectively. RESULTS: Ninety-two patients were included (83 in sinus, 9 in atrial fibrillation). 3DLV and 3DRV analyses were feasible in 72% and 55% of patients, respectively; however, they underestimated SV compared with Doppler by 2.6 ml (± 10.4) and 4.1 ml (± 15.4), respectively. Limits of agreement for 2D, 3DLV and 3DRV volumetric analysis techniques were large. CONCLUSIONS: 3DLV and 3DRV volumetric analyses appear feasible (obtainable) in the majority of mechanically ventilated ICU patients. Compared with the Doppler method, 3DLV and 3DRV volumetric analyses underestimate SV. The large limits of agreement between the methods also cast doubt on their comparability. Given the scenarios in which SV analysis is required (e.g., assessment of cardiac performance), our study cautions against the use of 3D SV clinically.

Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation.

Biofilms are communities of microbial cells that are encased in a self-produced, polymeric matrix and are adherent to a surface. For several species of bacteria, an enhanced ability to form biofilms has been linked with an increased capability to produce exopolymers. To identify exopolymers of Bacillus subtilis that can contribute to biofilm formation, we transferred the genetic determinants that control exopolymer production from a wild, exopolymer-positive strain to a domesticated, exopolymer-negative strain. Mapping these genetic determinants led to the identification of gamma-poly-dl-glutamic acid (gamma-PGA) as an exopolymer that increases biofilm formation, possibly through enhancing cell-surface interactions. Production of gamma-PGA by Bacillus subtilis was known to be dependent on the two-component regulator ComPA; this study highlighted the additional dependence on the DegS-DegU, DegQ and SwrA regulator proteins. The inability of the domestic strain of B. subtilis to produce gamma-PGA was mapped to two base pairs; a single base pair change in the promoter region of degQ and a single base pair insertion in the coding region of swrA. Introduction of alleles of degQ and swrA from the wild strain into the domestic strain was sufficient to allow gamma-PGA production. In addition to controlling gamma-PGA production, ComPA and DegSU were also shown to activate biofilm formation through an as yet undefined pathway. The identification of these regulators as affecting gamma-PGA production and biofilm formation suggests that these processes are regulated by osmolarity, high cell density and phase variation.

Environmental signals and regulatory pathways that influence biofilm formation.

In nature, bacteria often exist as biofilms. Here, we discuss the environmental signals and regulatory proteins that affect both the initiation of bacterial biofilm formation and the nature of the mature biofilm structure. Current research suggests that the environmental signals regulating whether bacterial cells will initiate a biofilm differ from one bacterial species to another. This may allow each bacterial species to colonize its preferred environment efficiently. In contrast, some of the environmental signals that have currently been identified to regulate the structure of a mature biofilm are nutrient availability and quorum sensing, and are not species specific. These environmental signals evoke changes in the nature of the mature biofilm that may ensure optimal nutrient acquisition. Nutrient availability regulates the depth of the biofilm in such a way that the maximal number of cells in a biofilm appears to occur at suboptimal nutrient concentrations. At either extreme, nutrient-rich or very nutrient-poor conditions, greater numbers of cells are in the planktonic phase where they have greater access to the local nutrients or can be distributed to a new environment. Similarly, quorum-sensing control of the formation of channels and pillar-like structures may ensure efficient nutrient delivery to cells in a biofilm.

Role of the Escherichia coli Tat pathway in outer membrane integrity.

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.

Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays.

Biofilms are structured communities of cells that are encased in a self-produced polymeric matrix and are adherent to a surface. Many biofilms have a significant impact in medical and industrial settings. The model gram-positive bacterium Bacillus subtilis has recently been shown to form biofilms. To gain insight into the genes involved in biofilm formation by this bacterium, we used DNA microarrays representing >99% of the annotated B. subtilis open reading frames to follow the temporal changes in gene expression that occurred as cells transitioned from a planktonic to a biofilm state. We identified 519 genes that were differentially expressed at one or more time points as cells transitioned to a biofilm. Approximately 6% of the genes of B. subtilis were differentially expressed at a time when 98% of the cells in the population were in a biofilm. These genes were involved in motility, phage-related functions, and metabolism. By comparing the genes differentially expressed during biofilm formation with those identified in other genomewide transcriptional-profiling studies, we were able to identify several transcription factors whose activities appeared to be altered during the transition from a planktonic state to a biofilm. Two of these transcription factors were Spo0A and sigma-H, which had previously been shown to affect biofilm formation by B. subtilis. A third signal that appeared to be affecting gene expression during biofilm formation was glucose depletion. Through quantitative biofilm assays and confocal scanning laser microscopy, we observed that glucose inhibited biofilm formation through the catabolite control protein CcpA.

Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation.

The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

mRNA secondary structure modulates translation of Tat-dependent formate dehydrogenase N.

Formate dehydrogenase N (FDH-N) of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized in an (alphabetagamma)3 configuration. The FdnG subunit carries a Tat-dependent signal peptide, which localizes the protein complex to the periplasmic side of the membrane. We noted that substitution of the first arginine (R5) in the twin arginine signal sequence of FdnG for a variety of other amino acids resulted in a dramatic (up to 60-fold) increase in the levels of protein synthesized. Bioinformatic analysis suggested that the mRNA specifying the first 17 codons of fdnG forms a stable stem-loop structure. A detailed mutational analysis has demonstrated the importance of this mRNA stem-loop in modulating FDH-N translation.

Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis.

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

Behaviour of topological marker proteins targeted to the Tat protein transport pathway.

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding beta-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat-dependent membrane protein complexes.

Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis.

The Escherichia coli Tat apparatus is a membrane-bound protein translocase that serves to export folded proteins synthesized with N-terminal twin-arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side-chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat-dependent protein export activity and thus represented residues essential for TatC function. Cross-complementation experiments with class I mutants showed that co-expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co-existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N-tail and first cytoplasmic loop regions of the TatC protein.