- Volume 149, Issue 4, 2003

The lipopolysaccharide (LPS) molecules of Shigella flexneri 2a have O antigen (Oag) polysaccharides with two modal chain length distributions. The chromosomal wzz SF gene results in short (S) type Oag chains [11–17 Oag repeat units (RUs)], and the pHS-2 plasmid-located wzz pHS2 gene results in very long (VL) type Oag chains (>90 Oag RUs). S. flexneri wzz SF mutants are unable to form plaques on HeLa cell monolayers and F-actin comet tails, indicating that IcsA/VirG function in actin-based motility (ABM) is defective. An S. flexneri wzz SF wzz pHS2 double mutant had LPS with relatively short, random length Oag chains and, paradoxically, was able to form plaques and F-actin comet tails. The influence of Oag modal chain length distribution on virulence and related properties was investigated using complementation with different wzz genes. WzzO139 from Vibrio cholerae O139 and WzzST from Salmonella enterica serovar Typhimurium were fully functional in Shigella flexneri, resulting in LPS with either very short (VS) type Oag chains (2–7 Oag RUs) or long (L) type Oag chains (19–35 RUs), respectively. In the absence of VL-type Oag chains, the VS-, S- and L-type Oag chains were permissive for plaque and F-actin comet tail formation. However, in the presence of LPS with VL-type Oag chains, the VS- and S-type Oag chains but not the L-type Oag chains were permissive for plaque and F-actin comet tail formation. These data, and the results of a previous investigation, show that IcsA function in ABM requires LPS Oag chains with at least two but less than 18 RUs when VL-type Oag chains are co-expressed on the cell surface. However, in the absence of the VL-type Oag chains, LPS Oag chains with at least two but less than 90 RUs are able to support IcsA function in ABM. Indirect immunofluorescence staining of IcsA on the cell surface of the S. flexneri strains did not correlate with the observed effect of Oag chain length on plaque and F-actin comet tail formation. However, when intracellular bacteria lacking VL-type Oag chains were examined, an inverse correlation between Oag modal chain length and detection of IcsA was observed, i.e. staining decreased with increased modal length. It is hypothesized that Oag chains can mask IcsA and interfere with its function in ABM, and a model is presented to explain how LPS Oag and IcsA may interact. It is suggested that S. flexneri 2a has evolved to synthesize LPS with two Oag modal chain lengths, as S-type Oag chains allow IcsA to function in ABM in the presence of VL-type Oag chains that confer resistance to serum.

In the study of differential gene expression of Cryptococcus neoformans, a transcript of COX1 (cytochrome oxidase c subunit 1) was identified in a serotype A strain. The transcript was upregulated at 37 °C compared to 30 °C and expressed by yeasts infecting the central nervous system. Northern analysis of COX1 from the serotype A strain revealed two polycistronic transcripts, a temperature-upregulated 2·3 kb transcript and a 1·9 kb transcript that was not affected by temperature. In contrast, COX1 in a serotype D strain showed only a 1·9 kb polycistronic transcript plus a 1·6 kb monocistronic message, and temperature had no effect on the transcripts. The sequence of COX1 revealed similar coding regions between the two strains, but the serotype D strain had five introns whereas no introns were found in the serotype A strain. The serotype D strain had reduced growth rates compared to the serotype A strain at 37 °C, but in an AD hybrid strain the serotype D COX1 gene could support efficient high temperature growth. These studies have revealed mitochondrial molecular differences between serotype A and D strains which show evolutionary divergence. It will be important to determine whether differences in mitochondrial structure and function can influence cryptococcosis.

The genetic organization and sequences of the group II capsule gene cluster of Aeromonas hydrophila PPD134/91 have been determined previously. The purified capsular polysaccharides can increase the ability of avirulent strain PPD35/85 to survive in naive tilapia serum but have no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells. In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was examined by electron microscopy and genetic analysis. Ten strains were found to produce group II capsules. A PCR detection system was developed to identify two types of group II capsules (IIA and IIB) based on their genetic organization in the region II gene clusters. Group IIA capsules in the authors' collection of A. hydrophila strains are mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules are found in the O : 21 and O : 27 serogroups. The presence of group II capsules in A. hydrophila strongly correlates with the serum and phagocyte survival abilities (seven out of ten strains). The results indicate that the authors' PCR detection system can constitute a reliable assay for the classification of group II capsules in A. hydrophila.

Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1·5-fold in the presence of bile salts and was decreased 50- to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxR-independent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0·2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation.

Micro-organisms used during the production of fermented foods are subjected to several abiotic stresses. Microbial survival during these processes strongly depends on the ability of the cells to adapt and become more tolerant to the environmental conditions. Cultivation of Lactobacillus amylovorus DCE 471, a potential strain for use during type II sourdough fermentations, at low temperatures, unfavourable pH and high salt concentrations resulted in biphasic growth patterns. In addition, two separate bacteriocin peaks, as well as a dramatic change in cellular morphology, were observed. In general, an increase of the specific bacteriocin production occurred during the second growth phase. Finally, the observed sugar consumption profiles were affected by the applied fermentation temperature. Moreover, the highest bacteriocin activity occurred during maltose consumption at a low constant temperature of 28 °C and a constant pH of 5·4. Plate counts from both growth phases revealed the existence of two colony types. Irregular colonies were found to outnumber smoother colonies during the first growth phase, while the second growth phase was characterized by a greater number of smooth colonies. Electron microscopy was used to investigate the observed morphological switch at the single-cell level. Single, rod-shaped cells changed into elongated cells that grew in chains. Colony and cell morphology changes coincided with the biphasic growth pattern.