Bacterial biofilms have gained increasing visibility in recent years as a ubiquitous form of survival for microorganisms in myriad environments. A number of in vivo models exist for the study of biofilms in the setting of medically relevant implanted foreign bodies. Growing evidence has demonstrated the presence of bacterial biofilms in the setting of a number of chronic wound states including pressure sores, diabetic foot ulcers, and venous stasis ulcers. Here we present a novel murine cutaneous wound system that directly demonstrates delayed reepithelialization caused by the presence of a bacterial biofilm. We established biofilms using either Staphylococcus aureus or Staphylococcus epidermidis in splinted cutaneous punch wounds created on the backs of normal C57Bl6/J mice. Wound reepithelialization was significantly delayed by bacterial biofilms. This effect was specifically dependent on the ability of the bacteria to form biofilms as demonstrated by exogenous administration of biofilm inhibiting peptides and the use of mutant Staphylococcus spp. deficient in biofilm formation. This represents the first direct evidence for the effect of bacterial biofilms on cutaneous wound healing.

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

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One of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing. In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane. Traditional methods of distinguishing between these two locations have relied on extensive, time-consuming biochemical studies. The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids. This numerical, sequence-based approach is usually seen as a first approximation pending more reliable biochemical data. The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved. We have optimized a statistical approach to protein location determination. Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method. We also examine the biological implications of the success of this technique.Keywords: computer modeling; discriminant analysis; hydropathy; membrane proteins; statistical methods Experimental methods of determination of subcellular protein location are accurate but time-consuming. Hydropathy analysis (Kyte & Doolittle, 1982) has often been used to deduce subcellular localization of proteins in the absence of experimental data. However, although visual inspection of hydropathy plots can be useful in predicting the topology of known integral membrane proteins, it is ineffective as an accurate predictor of the location of a protein.To discriminate between integral and peripheral membrane proteins, Klein et al. (1985) generated a single number, maxH, the average hydropathy of the most hydrophobic protein segment of a given length for a given protein using a given hydropathy scale. In the interest of clarity, we will refer to this number as the "maxH value," while using the term "maxH segment" to refer to the hydrophobic peptide segment to which it belongs. This was then applied to a set of known integral and peripheral membrane proteins in a training set. A discriminator function was generated that assigned a probability of being an integral membrane protein to a given value of maxH. This function was then used to analyze a similar set of known proteins, the tester set. It was determined that the Kyte-Doolittle hydropathy scale and a window length of 17 residues gave the best resolution of membrane and soluble proteins in the tester set. We have found that the method used by Klein et al. (1985) is still generally useful, but that the actual functions provided in their paper are not, having been derived at a time when very few proteins were both sequenced and characterized. ResultsWe discovered the need for a new discriminator when we attempted to apply the functions described by Klei...

Most extracytoplasmic proteins are synthesized with an N‐terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N‐terminus of alkaline phosphatase. These changes do not disrupt an N‐terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N‐terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane‐embedded export machinery.