DNA sequencing upstream of the Salmonella enterica serovar Typhi pilV and rci genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139-146, 1997) identified a further 10 pil genes apparently forming a pil operon. The product of the pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole pil operon or with an insertion mutation in the pilS gene were constructed, as was a strain in which the pilN to pilV genes were driven by the tac promoter. The pil Earlier, it was reported that the major pathogenicity island of Salmonella enterica serovar Typhi, which is ca. 118 kb in size (11), contained pilV and rci genes, which were cloned and sequenced (22). The Rci gene product was shown to be a site-specific recombinase, active to invert DNA in the C-terminal region of the pilV gene, so that two PilV proteins could be synthesized. Comparisons with database sequences indicated that the two possible pilV genes might code for pilus-tip adhesins, as the serovar Typhi PilV sequence was similar to that of PilV proteins encoded by the Escherichia coli R64 plasmid. In R64-bearing strains, different PilV proteins, borne on type IV pili, select various recipients in liquid mating (the R64-bearing cell is the donor) (10). Both serovar Typhi PilV proteins were seen when the two pilV genes were transcribed from the T7 promoter. The discovery of the serovar Typhi pilV and rci genes in the ca. 118-kb pathogenicity island (henceforth in this work termed the large pathogenicity island) suggested that serovar Typhi might synthesize thin pili belonging to the type IV pilin family (9). As type IV pili, encoded in a Vibrio cholerae pathogenicity island (7, 8) are used by V. cholerae as mediators of adhesion to human cells (13, 18), it was of interest to ask (i) if serovar Typhi also synthesizes type IV pili and (ii) if such pili are important in adherence to or invasion of human intestinal cells. These topics are the subject of this paper. MATERIALS AND METHODSMaterials. All reagents were of molecular biology grade. Enzymes active on DNA were obtained from either GibcoBRL or Boehringer Mannheim and were used as directed by the suppliers. 5-Bromo-4-chloro-3-indolyl-␤-D-galactopyranoside and isopropyl-␤-D-thiogalactopyranoside were purchased from Amersham. Anti-mouse immunoglobulin G (from sheep), conjugated with horseradish peroxidase, was from Amersham. Phosphatase-labeled goat anti-mouse immunoglobulin G (heavy and light chains) was purchased from KPL Laboratories. p-Nitrophenyl phosphate tablets were from Sigma. Bio-Rad was the supplier of polyvinylidene difluoride membrane. Freund's adjuvant was from GibcoBRL.Strains and vectors. Serovar Typhi J341 (Ty2 Vi Ϫ ) (22) was the source of DNA for a cosmid bank (partially Sau3AI-cut DNA in BamHI-cut pHC79), which was probed with 32 P-labeled total DNA (including the virulence plasmid pSLT) of (wild-type,...

There has been little written on the penetration of light through soil. In this review, we attempt to collate most of the work that has been published on this topic in order to stimulate further research and to clarify the often‐confused literature. Light penetration can be measured directly with, for example, a spectroradiometer, or indirectly by using germination of light‐sensitive seeds or the presence of growing algae as bioindicators. Although the penetration of light through soil is greatly affected by factors such as soil moisture content and particle size and colour, it generally appears that physiologically and ecologically significant amounts of light rarely penetrate more than 4–5 mm through the soil, and may often penetrate much less than this. Any penetration beyond 10 mm would generally not be significant, especially as most soils are covered with litter, algae or lichens, or are shaded from direct sunlight. However, for some light‐stimulated geo‐tropic responses of roots, which can be sensitive to very low fluences, the penetration of light to greater depths could well be significant. The role of light in soil in directing root growth is also discussed.

Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes (27). MDH is a tetrameric enzyme located in the periplasm that contains pyrroloquinoline quinone (PQQ) as the prosthetic group and also contains Ca2+ (37,40). PQQ was first identified as the prosthetic group of MDH and is now also known to be the prosthetic group of a few other bacterial dehydrogenases that oxidize alcohols or sugars (9). The biosynthetic pathway of PQQ has not yet been determined, but the biosynthetic precursors are known to be tyrosine and glutamate (19).

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.