Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont.

The cell wall amidase AmiB is essential for Pseudomonas aeruginosa cell division, drug resistance and viability.

The physiological function of cell wall amidases has been investigated in several proteobacterial species. In all cases, they have been implicated in the cleavage of cell wall material synthesized by the cytokinetic ring. Although typically non-essential, this activity is critical for daughter cell separation and outer membrane invagination during division. In Escherichia coli, proteins with LytM domains also participate in cell separation by stimulating amidase activity. Here, we investigated the function of amidases and LytM proteins in the opportunistic pathogen Pseudomonas aeruginosa. In agreement with studies in other organisms, (Pa) AmiB and three LytM proteins were found to play crucial roles in P. aeruginosa cell separation, envelope integrity and antibiotic resistance. Importantly, the phenotype of amidase-defective P. aeruginosa cells also differed in informative ways from the E. coli paradigm; (Pa) AmiB was found to be essential for viability and the successful completion of cell constriction. Our results thus reveal a key role for amidase activity in cytokinetic ring contraction. Furthermore, we show that the essential function of (Pa) AmiB can be bypassed in mutants activated for a Cpx-like envelope stress response, suggesting that this signaling system may elicit the repair of division machinery defects in addition to general envelope damage.

High salt stress in Bacillus subtilis: involvement of PBP4* as a peptidoglycan hydrolase.

The study was focused on the role of the penicillin binding protein PBP4* of Bacillus subtilis during growth in high salinity rich media. Using pbpE-lacZ fusion, we found that transcription of the pbpE gene is induced in stationary phase and by increased salinity. This increase was also corroborated at the translation level for PBP4* by western blot. Furthermore, we showed that a strain harboring gene disruption in the structural gene (pbpE) for the PBP4* endopeptidase resulted in a salt-sensitive phenotype and increased sensitivity to cell envelope active antibiotics (vancomycin, penicillin and bacitracin). Since the pbpE gene seems to be part of a two-gene operon with racX, a racX::pRV300 mutant was obtained. This mutant behaved like the wild-type strain with respect to high salt. Electron microscopy showed that high salt and mutation of pbpE resulted in cell wall defects. Whole cells or purified peptidoglycan from WT cultures grown in high salt medium showed increased autolysis and susceptibility to mutanolysin. We demonstrate through zymogram analysis that PBP4* has murein hydrolyze activity. All these results support the hypothesis that peptidoglycan is modified in response to high salt and that PBP4* contributes to this modification.

Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model.

A rat central venous catheter (CVC) infection model was used to assess the importance of the proteinacious autolysin (AtlE) and the polysaccharide intercellular adhesin (PIA) in the pathogenesis of Staphylococcus epidermidis CVC-associated infection. Wild-type (wt) S. epidermidis O-47 was significantly more likely to cause a CVC infection than was either of the isogenic mutant strains (AtlE-negative [O-47mut1] or PIA-negative [O-47mut2]). Bacteria were retrieved from the explanted catheters of 87.5% of rats inoculated with S. epidermidis O-47, compared with 25% of rats challenged with either S. epidermidis O-47mut1 or O-47mut2 (P=.007). Peripheral bacteremia was documented in 75% of rats challenged with S. epidermidis O-47, compared with 12.5% and 25% challenged with O-47mut1 and O-47mut2, respectively (P=.009). Metastatic disease was more common in rats inoculated with wt S. epidermidis, compared with AtlE- or PIA-deficient mutants. These results confirm the importance of initial adherence, associated with AtlE, and biofilm production, mediated by PIA, in the pathogenesis of S. epidermidis experimental CVC infection.

Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A.

An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.