The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.

The molecular weight distribution of the asphaltene fractions of two types of crude oils from two different Italian fields (samples 1 and 2) was investigated. The analytical tools used to perform these analyses were matrix assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) mass spectrometry. After observing that the use of the matrix (as well as the addition of Ag+) did not improve the quality of the data compared to that obtained in LDI conditions, all further measurements were performed with the latter technique. Operating under usual conditions of laser power and delay time, a very low resolution was observed, showing only macroscopic differences between the two samples in the molecular weight distribution of the different components. An accurate study on the possible reasons of this undesirable behavior indicates that it can originate from space charge phenomena occurring either in the ion source region or during the flight. A valid parameterization of the delay time and the laser power allowed higher quality spectra to be obtained. Surface-enhanced laser desorption ionization (SELDI) measurements were also performed using normal phase (silica) as the sample holder surface. Under these conditions, better results are obtained, proving that the sample-surface interaction is important to achieve, by means of laser irradiation, a homogeneous set of product ions. Both asphaltene samples were fractionated in five subfractions by gel-permeation chromatography (GPC) to obtain a better separation of the molecular weight distributions; the related spectra confirmed these findings. By using different approaches, relevant and reproducible differences between the asphaltene fractions of the two oil samples were observed.

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5' portion of the srfA operon affect all three of these processes, whereas mutations in the 3' portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colinearity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5' region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.

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A large operon-type structure has been located between the g/tA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.Keywords : Bacillus subtilis, peptide synthase operon, racemase Micro-organisms produce a large number of peptides, generally as secondary metabolites, which have many important activities varying from antibacterial to antiviral or antifungal, and from anticarcinogenic to immunosuppressive. Regardless of their function, these peptides are synthesized either using the usual ribosomal system or through the action of complex multi-enzyme systems known as peptide synthases.It is now clear that although their products can vary considerably in length and amino acid composition, all peptide synthases studied so far share remarkable similarity in their structural organization and mechanism of action (Kleinkauf & von Dohren, 1990; Cosmina e t al., 1993). They are in fact organized into structural domains, each of which is responsible for the recognition and binding of a specific amino acid. Peptide synthesis takes place through subsequent reactions of thioester cleavage and amide bond formation.Bacillzrs szxbtilis produces a number of peptides and lipopeptides, both linear and cyclic (Zuber etal., 1993). Of these, only surfactin has been thoroughly characterized at the genetic level (Cosmina e t al., 1993; van Sinderen e t al., 1993). From gene sequence analysis and a variety of biochemical studies, it was found that surfactin synthase is a multi-subunit enzyme complex in which there are seven structural domains, corresponding to the number of amino acids present in surfactin.In this communication we report the identification in the B. mbtilis chromosome of a large operon encoding a new peptide synthase.The 15.4 kb fragment present in A clone AB21 (Fig. l) (Kunst & Devine, 1991), two additional A clones (A1A and A4B) were identified by D N A : DNA hybridization, the inserts of which overlap with the chromosomal fragment carried by AB21 (Fig. 1). Finally, an additional 4.3 kb overlapping fragment was isolated by chromosome walking (Glaser et a/., 1993) after cloning the AB21 HindIII-AcyI fragment into the suicide vector pDIA5304. Altogether, the three A clones and the pDIA5304 derivative contained an uninterrupted chromosomal region of approximately 25 kb.The identity of the 25 kb region carried by the A and plasmid vectors with the corresponding chromosomal area was confirmed by both restriction enzyme and Southern blot analyses. In particular, when AB21, A4B and A1A were digested with EcoRI and NotI, fragments of identical size were identified on an agarose gel. Furthermore, the large 3.0 kb EcoRI fragment of the three clones ( Fig. 1) hybridized with an intern...