Streptococcus mitis is the closest relative of the major human pathogen S. pneumoniae. The 2,15 Mb sequence of the Streptococcus mitis B6 chromosome, an unusually high-level beta-lactam resistant and multiple antibiotic resistant strain, has now been determined to encode 2100 genes. The accessory genome is estimated to represent over 40%, including 75 mostly novel transposases and IS, the prophage φB6 and another seven phage related regions. Tetracycline resistance mediated by Tn5801, and an unusual and large gene cluster containing three aminoglycoside resistance determinants have not been described in other Streptococcus spp. Comparative genomic analyses including hybridization experiments on a S. mitis B6 specific microarray reveal that individual S. mitis strains are almost as distantly related to the B6 strain as S. pneumoniae. Both species share a core of over 900 genes. Most proteins described as pneumococcal virulence factors are present in S. mitis B6, but the three choline binding proteins PcpA, PspA and PspC, and three gene clusters containing the hyaluronidase gene, ply and lytA, and the capsular genes are absent in S. mitis B6 and other S. mitis as well and confirm their importance for the pathogenetic potential of S. pneumoniae. Despite the close relatedness between the two species, the S. mitis B6 genome reveals a striking X-alignment when compared with S. pneumoniae.

PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.

The largest of the fragments produced by AluI digestion of phi X174 RFI DNA comprises genes E and J as well as parts of genes D and F. This DNA fragment (1007 bp) was cloned into the lac z' gene of plasmid pUR222. In the recombinant plasmid pUH12, transcription of the phi X174 genes is controlled by the lac p-o region. Induction of the cloned genes by addition of the lac inducer, IPTG, resulted in lysis of the bacteria. Cloning of the corresponding AluI-fragment from phi X174am3 DNA, carrying an amber mutation in gene E, showed that the expression of this gene alone is sufficient to trigger cell lysis. The time interval between the addition of IPTG and the onset of lysis depended on the concentration of the inducer, however, the rate of lysis was similar at all IPTG concentrations used.

The lysis genes of the Lactobacillus gasseri bacteriophage adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage LC3. Significant homology with the N termini of known muramidases suggests that adh lysin acts by a similar catalytic mechanism. In E. coli, the adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage 29. Concomitant expression of the combined holin and lysin functions of adh in E. coli, however, did not result in efficient cell lysis.Large Escherichia coli phage in general appear to encode at least two lysis functions, a murein hydrolase, required for destruction of the peptidoglycan, and a protein termed holin which permits access of the lytic enzyme to the periplasm (for a review, see reference 78). In the case of bacteriophage lambda, oligomerization of the holin (protein S) in the inner membrane of E. coli apparently leads to formation of a nonspecific lesion through which the lambda transglycosylase is released to the periplasm at the end of the vegetative cycle (80). The expression of the S gene and thus the kinetics of formation of the S-dependent hole in the inner membrane is tightly controlled at the transcriptional (35) and at the translational level (7, 53), as well as posttranslationally by virtue of two S-encoded polypeptides with opposing functions (6, 70). Holin functions have also been attributed to the products of P22 gene 13 (58), to phage 21 gene S (9), and recently to protein 14 of the Bacillus subtilis phage 29 (70), the first holin identified from a phage of gram-positive bacteria. Large phage therefore appear to pursue an evolutionarily conserved lysis pathway.In phage lambda, P22, 21, and 29, the genes encoding the corresponding holins and murein hydrolases are arranged identically. The holin gene in all cases precedes the gene encoding the murein hydrolase and overlaps at least with its ribosome-binding site (9,13,30,58). With the exception of the products of the lambda S and P22 13 genes, which are nearly identical (58), the known holins show no homology with each other (78). Since lambda S...

The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41,281 daltons) and EnvD (104,415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promoter without deleterious effects on the host.