Methicillin-resistant Staphylococcus saprophyticus isolates carrying staphylococcal cassette chromosome mec have emerged in urogenital tract infections.

Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed beta-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

The sigH gene sequence can subspeciate staphylococci.

In an evolutionarily conserved gene organization (syntenic region), the sigH gene shares exceptionally low homology among staphylococcal species. We analyzed the "positionally cloned" sigH sequences of 39 staphylococcal species. The topology of the SigH phylogenetic tree was consistent with that of 16S rRNA. Certain clinical isolates were successfully differentiated at the species level with the sigH sequence data set. We propose that the sigH gene is a promising molecular target in genotypic identification because it is highly discriminative in differentiating closely related staphylococcal species.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.

In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.

Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.

Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection.

Staphylococcus saprophyticus is a uropathogenic Staphylococcus frequently isolated from young female outpatients presenting with uncomplicated urinary tract infections. We sequenced the whole genome of S. saprophyticus type strain ATCC 15305, which harbors a circular chromosome of 2,516,575 bp with 2,446 ORFs and two plasmids. Comparative genomic analyses with the strains of two other species, Staphylococcus aureus and Staphylococcus epidermidis, as well as experimental data, revealed the following characteristics of the S. saprophyticus genome. S. saprophyticus does not possess any virulence factors found in S. aureus, such as coagulase, enterotoxins, exoenzymes, and extracellular matrix-binding proteins, although it does have a remarkable paralog expansion of transport systems related to highly variable ion contents in the urinary environment. A further unique feature is that only a single ORF is predictable as a cell wall-anchored protein, and it shows positive hemagglutination and adherence to human bladder cell associated with initial colonization in the urinary tract. It also shows significantly high urease activity in S. saprophyticus. The uropathogenicity of S. saprophyticus can be attributed to its genome that is needed for its survival in the human urinary tract by means of novel cell wall-anchored adhesin and redundant uro-adaptive transport systems, together with urease.

A 32 kb nucleotide sequence from the region of the lincomycin-resistance gene (22 degrees-25 degrees) of the Bacillus subtilis chromosome and identification of the site of the lin-2 mutation.

A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22 degrees to 25 degrees on the Bacillus subtilis chromosome, was determined. Among 32 putative ORFs identified, four [lipA for lipase, natA, natB and yzaE (renamed yccK)] have already been reported, although the functions of NatA, NatB and YccK remain to be characterized. Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, alpha-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B. subtilis. The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon. The products of 14 ORFs exhibited considerable or limited similarity to known proteins. The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains. The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing. This site is located between a putative promoter and the SD sequence of ImrA (yccB) [a putative repressor of the lmr operon, which consists of lmrA and lmrB (yccA)]. LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin.

Multidrug resistant phenotype of Bacillus subtilis spontaneous mutants isolated in the presence of puromycin and lincomycin.

Spontaneous mutants were isolated by growing Bacillus subtilis 168 in the presence of high concentrations of puromycin and lincomycin. These mutants showed increased resistance to several drugs other than these two drugs. The ImrAB genes, which encode a transcriptional repressor and a drug efflux protein of the major facilitator superfamily, were involved in this phenotype. Northern hybridization analysis showed that the expression of ImrAB gene increased more than 30-fold. The following two types of mutations were found to be responsible for the multidrug resistant phenotype: (i) a nucleotide replacement in the region between the promoter and initiation codon of ImrA and (ii) nucleotide replacements that resulted in amino acid replacements in the LmrA protein. The results indicate that LmrB is a multidrug resistant protein and that LmrA is a repressor, which autogenously represses the transcription of the ImrAB operon.

Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study.

To analyse the proteome of Bacillus subtilis extracellular proteins, extracellular protein samples were prepared from culture media (minimal medium containing 0.4% glucose) of parental B. subtilis 168, a secA-temperature sensitive mutant and an ffh conditional mutant, and examined by two-dimensional gel electrophoresis. Approximately 100 to 110 spots were visualized in a gel of B. subtilis 168 extracellular proteins. Over 90% and 80% of these disappeared in the absence of SecA and Ffh, respectively. Thirty-eight obvious spots on the gel of the B. subtilis 168 preparation were selected and compared with spots obtained under SecA- or Ffh-deficient conditions. The appearance of 36 of these 38 spots depended on SecA and Ffh. Nineteen additional extracellular proteins were detected in cultures maintained in cellobiose, maltose and soluble starch. Among 23 proteins of which the N-terminal amino acid sequences were determined, 17 were extracellular proteins having signal peptides in their precursor form. Two membrane proteins, Yfnl and YflE, were cleaved behind 226Ala-Tyr-Ala228 and 213Ala-Leu-Ala215, respectively, and of which products seemed to be liberated into the culture medium. The production of Yfnl and YflE were also dependent on SecA and Ffh. These results indicate that most extracellular proteins target to and translocate across the cytoplasmic membrane by co-operation between the signal-recognition particle and Sec protein-secretion pathways. In contrast, a spot for Hag appeared independent from SecA and Ffh. Intracellular proteins Gap, SodA and KatA were identified in the extracellular protein samples. On the basis of these results and computer searches, it was predicted that B. subtilis produces 150 to 180 proteins extracellularly.

Lincomycin resistance mutations in two regions immediately downstream of the -10 region of lmr promoter cause overexpression of a putative multidrug efflux pump in Bacillus subtilis mutants.

We isolated 19 lincomycin-resistant Bacillus subtilis mutants by expressing lmrB encoding a putative multidrug efflux protein. Eighteen of the mutants altered at two regions (-3 to -1 and +15) immediately downstream of the -10 region of the lmr promoter increased lmr transcription in vivo and in vitro.

Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons: identification of its binding site and functional analysis of lmrB and yxaGH.

The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.