The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Increased thermal resistance and modification of the catalytic properties of a beta-glucosidase by random mutagenesis and in vitro recombination.

The bglB gene from Paenibacillus polymyxa was subjected to random mutagenesis mediated by error prone polymerase chain reaction amplification and DNA shuffling. After this treatment, mutant variants of the encoded beta-glucosidase with enhanced thermal resistance were selected. We identified five amino acid substitutions at four different positions of the sequence that increased the resistance of the enzyme to heat denaturation. Four of the mutations, H62R, M319V, M319I, and M361I, did not change the kinetic parameters of the enzyme. However, mutant N223Y, which caused only a marginal increase in thermoresistance, showed an 8-fold decrease in K(m). Copies of the bglB gene carrying each one of the individual mutations were recombined in vitro by DNA shuffling. As a result, we obtained an enzyme that simultaneously exhibited a 20-fold increase in heat resistance and an 8-fold increase in the catalytic efficiency. The structural basis of the properties conferred by the mutations was analyzed using homology-based structural models. The four mutations causing a more pronounced effect on thermoresistance were located in loops, on the periphery of the (alpha/beta)(8) barrel that conforms the structure of the protein. Mutation N223Y, which modifies the catalytic properties of the enzyme, was on one of the barrel beta-strands that shape the active center.

Regulation of alpha-helical coiled-coil dimerization in chicken skeletal muscle light meromyosin.

The dimerization specificity of the light meromyosin (LMM) domain of chicken neonatal and adult myosin isoforms was analyzed by metal chelation chromatography. Our results show that neonatal and adult LMMs associate preferentially, although not exclusively, as homodimeric coiled-coils. Using chimeric LMM constructs combining neonatal and adult sequences, we observed that a stretch of 183 amino acids of sequence identity at the N terminus of the LMM was sufficient to allow the adult LMM to dimerize in a non-selective manner. In contrast, sequence identity in the remaining C-terminal 465 amino acids had only a modest effect on the dimerization selectivity of the adult isoform. Sequence identity at the N terminus also promoted dimerization of the neonatal LMM to a greater degree than sequence identity at the C terminus. However, the N terminus had only a partial effect on the dimerization specificity of the neonatal sequence, and residues distributed throughout the LMM were capable of affecting dimerization selectivity of this isoform. These results indicated that dimerization preference of the neonatal and adult isoforms was affected to a different extent by sequence identity at a given region of the LMM.

The role of interhelical ionic interactions in myosin rod assembly.

Interhelical electrostatic interactions at specific heptad positions can regulate dimerization specificity of alpha-helical coiled-coils. We have analyzed 20 vertebrate myosin sequences from a variety of organisms and tissues in order to determine if interhelical ionic interactions correlate with the observed myosin dimerization specificity. We find that the sites for potential interhelical ion pairing are identical in virtually all sarcomeric myosins whether they form homo- or heterodimers. We also show that smooth muscle and non-muscle myosin rod sequences exhibit a different conserved pattern of potential interhelical ion pairing. These observations suggest that myosin rod residues involved in interhelical electrostatic interactions do not regulate dimerization specificity, but may contribute to the specific arrangements of myosin molecules that determine differences in the filament morphology of sarcomeric and non-sarcomeric muscles.

The evolution of the chicken sarcomeric myosin heavy chain multigene family.

This manuscript describes the chicken sarcomeric myosin heavy chain (MyHC) multigene family and how it differs from the sarcomeric MyHC multigene families of other vertebrates. Data is discussed that suggests the chicken fast MyHC multigene family has undergone recent expansion subsequent to the divergence of avians and mammals, and has been subjected to multiple gene conversion-like events. Similar to human and rodent MyHC multigene families, the chicken multigene family contains sarcomeric MyHC genes that are differentially regulated in developing embryonic, fetal, and neonatal muscles. However, unlike mammalian genes, chicken fast MyHC genes expressed in developing muscles are also expressed in mature muscle fibers as well. The potential significance of conserved and divergent sequences with the MyHC rod domain of five fast chicken isoforms that have been cloned and sequenced is also discussed.

The evolutionary relationship of avian and mammalian myosin heavy-chain genes.

Sequence comparisons of avian and mammalian skeletal and cardiac myosin heavy-chain isoforms are used to examine the evolutionary relationships of sarcomeric myosin multigene families. Mammalian fast-myosin heavy-chain isoforms from different species, with comparable developmental expression, are more similar to each other than they are to other fast isoforms within the same genome. In contrast, the developmentally regulated chicken fast isoforms are more similar to each other than they are to myosin heavy-chain isoforms in other species. Extensive regions of nucleotide identity among the chicken fast myosin heavy chains and in the mouse and rat alpha- and beta-cardiac myosin heavy-chain sequences suggest that gene-conversion-like mechanisms have played a major role in the concerted evolution of these gene families. We also conclude that the chicken fast myosin heavy-chain multigene family has undergone recent expansion subsequent to the divergence of birds and mammals and that both the developmental regulation and the specialization of myosin isoforms have likely developed independently in birds and mammals.

Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.

Gene conversions within the skeletal myosin multigene family.

Comparisons of the nucleotide sequences of the light meromyosin (LMM) region of developmentally regulated fast chicken myosin heavy chain (MHC) isoforms indicates that chicken MHC isoforms are more similar to each other than to MHC isoforms in other species. The sequence data provide evidence that gene conversion events have occurred recently among the isoforms. An embryonic (Cemb1) isoform and neonatal isoform have the most extensive regions of sequence identity. Similar gene conversion events are present in the rat alpha- and beta-cardiac MHCs, but were not obvious in the LMM of developmentally regulated fast human MHC isoforms. The data suggest that gene conversion events can play a significant role in the evolution of the MHC multigene families and that concerted evolution of the chicken multigene family occurred after the divergence of mammals and avians.