Summary Some plant‐beneficial pseudomonads can invade and kill pest insects in addition to their ability to protect plants from phytopathogens. We explored the genetic basis of O‐polysaccharide (O‐PS, O‐antigen) biosynthesis in the representative insecticidal strains Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 and investigated its role in insect pathogenicity. Both strains produce two distinct forms of O‐PS, but differ in the organization of their O‐PS biosynthesis clusters. Biosynthesis of the dominant O‐PS in both strains depends on a gene cluster similar to the O‐specific antigen (OSA) cluster of Pseudomonas aeruginosa. In CHA0 and other P. protegens strains, the OSA cluster is extensively reduced and new clusters were acquired, resulting in high diversity of O‐PS structures, possibly reflecting adaptation to different hosts. CHA0 mutants lacking the short OSA form of O‐PS were significantly impaired in insect virulence in Galleria injection and Plutella feeding assays. CHA0, PCL1391, and other insecticidal pseudomonads exhibited high resistance to antimicrobial peptides, including cecropins that are central to insect immune defense. Resistance of both model strains depended on the dominant OSA‐type O‐PS. Our results suggest that O‐antigen is essential for successful insect infection and illustrate, for the first time, its importance in resistance of Pseudomonas to antimicrobial peptides.

Freshly collected cerumen (dry form) suspended at a concentration of 3% in glycerol-sodium bicarbonate buffer showed bactericidal activity against some strains of bacteria tested. This suspension reduced the viability of Haemophilus influenzae, Escherichia coli K-12, and Serratia marcescens by more than 99%, whereas the viability of two Pseudomonas aeruginosa isolates, E. coli K-1, Streptococcus, and two Staphylococcus aureus isolates of human origin was reduced by 30 to 80%. The results support the hypothesis that cerumen functions to kill certain foreign organisms which enter the ear canal.Cerumen, commonly known as earwax, is secreted by both ceruminous and sebaceous glands. Two distinct forms of human cerumen, dry and wet, are associated with race and controlled by two autosomal alleles (10). The dry allele is predominant in Mongoloid populations of Asia and in American Indians, whereas the wet allele is found predominantly in Caucasian and Negro populations (1, 10). Earwax has been found to contain amino acids, fatty acids, neurostearic acid, cerotic acid, cholesterol, triglyceride, hexone bases, lysozyme, immunoglobulin, glycopeptide, copper, and other components, although differences in composition between the cerumen types have been described (6,7,9,15).The function of cerumen in protecting the ear against invasion of microorganisms has long been a subject of controversy. It has been suggested that cerumen is unable to prevent infection and that the rich nutrients of earwax support luxuriant growth of bacteria and fungi (3,8,13,14). On the other hand, it has been suggested that cerumen might have antimicrobial activity, although little evidence has been presented to support this contention (5, 9). Burtenshaw (2) extracted cerumen with either saline or an alcohol-ether solvent and showed that the saline extract promoted the growth of streptococci somewhat, whereas the alcohol-ether extract was inhibitory. However, the concentration of cerumen in the alcohol-ether extract used by this author was not specified. In this communication we will describe a potent antibacterial activity of cerumen suspended in buffer against certain strains of common bacteria which are often encountered in humans.Pooled cerumen was collected with a sterile earwax hook from 12 healthy individuals aged from 5 to 42, including males and females, and kept in a sterile bottle at 4°C. All cerumen belonged to the typical dry form, which appeared flaky or granular and yellowish white. The pooled cerumen was mixed well, weighed, and suspended in buffer (5% NaHCO3, pH 8.2, containing 30% glycerol) at a concentration of 3.5% (wt/vol). The cerumen-buffer mixture was homogenized by repeated passage through a series of needles ranging from 19 to 23 gauge. This procedure broke the cerumen into fine particles distributed evenly in buffer and resulted in a milky suspension. Cerumen suspensions at concentrations over 3.5% were unsatisfactory because all the cerumen remained in big particles even after prolonged homogenization. The cerumen was ster...

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL displays an apparently more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.