- Volume 148, Issue 5, 2002

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. The VirB complex, which is highly similar to conjugative DNA transfer apparatuses, is required for intracellular replication. A conserved NTP-binding domain in VirB4 suggests that one or both proteins couple energy by NTP hydrolysis to transport of putative effector molecule(s). Here it is shown that a mutant strain of B. abortus that contains an in-frame deletion in virB4 is unable to replicate in macrophages and survives in mice. Intracellular replication and virulence in mice are fully restored by expressing virB4 in trans, indicating that VirB4 is essential for intracellular replication and virulence in mice. An alteration within the NTP-binding region of VirB4 by site-directed mutagenesis abolished complementation of a virB4 mutant, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Intracellular replication was inhibited in wild-type B. abortus after introducing a plasmid expressing a mutant VirB4 altered in the NTP-binding region. The dominant negative phenotype suggests that VirB4 either functions as a multimer or interacts with some other component(s) necessary for intracellular replication. Wild-type B. abortus-containing phagosomes lack the glycoprotein LAMP-1, which is an indicator of the normal endocytic pathway. Mutant strains were found in phagosomes that co-localized with LAMP-1, indicating that VirB4 containing the intact NTP-binding region is essential for evasion of fusion with lysosomes.

Strains of Helicobacter pylori that contain the cag pathogenicity island (cag PAI) are associated with increased virulence and severe clinical outcomes. To evaluate the role of the cag island in infection, isogenic null mutations were generated in two clinical isolates (SS1 and Iris1) with distinct genetic backgrounds. When tested for their ability to establish infection in the stomach of CD1/SPF mice, at the early phase of infection, strains in which cagE, ORF528, ORF527 or ORF525 were inactivated showed a reduced capacity to initiate colonization compared to the wild-type strain. Strains with a mutation in the ORF524 gene were more efficient than the other mutants, but still less efficient than the wild-type strain. Mutation in the effector protein, CagA, which is injected into host cells and tyrosine-phosphorylated, did not change the colonization efficiency. In conclusion, all cag genes analysed, with the exception of the effector protein, CagA, influenced the early phase of colonization in the mouse model of infection. These results suggest that the structure of the H. pylori secretion apparatus itself is involved in this process.

YopQ in Yersinia enterocolitica (YopK in Yersinia pseudotuberculosis) is a type III secreted protein required for virulence of yersiniae. In this study YopQ expression, secretion and nucleotide sequences of the corresponding yopQ gene from different yersinia strains were analysed. The cytosolic accumulation differed significantly among serotypes of Y. enterocolitica. These differences might be attributable to variations in the nucleotide sequence and their consequences on mRNA secondary structure. An mRNA signal hypothesis has been proposed for YopQ, predicting the coupling of translation and secretion via an mRNA signal. This hypothesis claims a strictly co-translational secretion of YopQ without its intracellular accumulation. The presence of YopQ in the cytosol, even with a closed secretion apparatus, is demonstrated. Moreover, post-translational secretion of YopQ could be demonstrated. These findings do not support the mRNA signal hypothesis for co-translational secretion.

Colonization of the human nasopharynx is a feature of some species of Neisseria, and is a prerequisite of invasive meningococcal disease. The likelihood of colonization by Neisseria meningitidis varies widely between humans, and very few develop invasive disease. Explants of nasal mucosa derived from adult patients with non-allergic nasal obstruction were infected experimentally with Neisseria spp. At intervals over 18 h incubation, washed explants were homogenized, and viable bacteria were counted. To estimate bacterial invasion of mucosa, explants were exposed to 0·25% sodium taurocholate for 30 s prior to homogenization. N. meningitidis was recovered from the mucosa and the organism invaded and replicated within the tissue, in contrast to N. lactamica and N. animalis (n=9, P<0·008). N. meningitidis isolates of clones ET-5, ET-37 and lineage III were recovered from and invaded tissue, but strains of clones A4, A:subgroup I, A:subgroup III and A:subgroup IV-1 did not invade (n=6). To measure host variation, survival of N. meningitidis within nasal mucosa of 40 different human donors was measured. Intra-class correlation of replicates was 0·97, but the coefficient of variation of recovered viable counts was 1335% after 4 h and 77% after 18 h incubation. It is concluded that the distinctive colonization and disease potential of Neisseria spp. may be partly a consequence of their ability to invade and survive within human nasopharyngeal mucosa, but that this is influenced greatly by genetic or environmental factors operating on the host mucosa. This is consistent with the unpredictable epidemiology of meningococcal disease.

The expression of a wide variety of physiological functions in many bacterial species is modulated by quorum sensing, a population-dependent signalling mechanism that involves the production and detection of extracellular signalling molecules. The genome sequence of Campylobacter jejuni NCTC 11168 contains a gene encoding an orthologue of LuxS, which is required for autoinducer-2 (AI-2) production in other bacterial species, but does not contain genes predicted to encode any known acyl-homoserine lactone synthetase. This study demonstrates that C. jejuni produces functional AI-2 activity through the ability of cell-free extracts to specifically induce bioluminescence in Vibrio harveyi BB170, a reporter strain for quorum-sensing system 2. Production of this signalling compound was shown to be dependent upon the product of the C. jejuni luxS gene (Cj1198). While the luxS mutant showed comparable growth rate, resistance to oxidative stress and ability to invade Caco-2 cell monolayers to the parental strain, it exhibited decreased motility haloes in semisolid media, suggesting a role for quorum sensing in the regulation of motility.

Differential fluorescence induction technology was used to identify promoters of Streptococcus pneumoniae genes that are expressed during lung infection of the mouse. Among the promoter clones that were identified multiple times was the psa promoter, which drives expression of the psaBCA operon. These genes have been identified previously and shown to encode a manganese permease system as well as play a role in the virulence of this organism. Mutations in psaB, psaC or psaA result in growth limitation in low manganese. The expression of the psa operon was examined in vivo and the virulence of deletion mutants of psaB, psaC, psaA and psaBCA was assessed in four different animal models of infection. The psa promoter was induced more than ten-fold in vivo using an intraperitoneal chamber implant model. The psaB, psaC and psaA mutants were completely attenuated in systemic, respiratory tract and otitis media infections. In addition, these mutants were unable to grow in an implanted peritoneal chamber, but growth was restored by the addition of manganese to the chambers.

The aim of this work was to characterize a 2·4 kb randomly amplified polymorphic DNA (RAPD) fragment described as a marker for a phylogenetic group of Streptococcus agalactiae strains significantly associated with neonatal meningitis. This fragment was analysed by cloning and sequencing, and showed that two types of tRNA gene cluster flank the 3′ end of the rRNA operons in S. agalactiae strains. Both types of tRNA gene cluster act as markers for phylogenetic subgroups of strains within the species. One type could be used to distinguish two of the three virulent intraspecies subgroups to which most of the S. agalactiae strains able to invade the central nervous system of neonates belong. This raises the possibility that there is a link between these tRNA genes and the virulence of the bacterium. Based on this analysis, PCR primers were designed to determine whether S. agalactiae strains are likely to belong to lineages of organisms in which most of the highly virulent strains isolated from cerebrospinal fluid cluster. In addition, this work demonstrated that RAPD can be used to detect novel particularities within intraspecies variants of pathogens.

RofA-like protein (RALP) type regulators have been shown to exist in different forms in group A streptococci (GAS) and to regulate the expression of important bacterial adhesins. This study shows that the vast majority of strains from different GAS M serotypes carried a rofA virulence regulator gene in their genome and that this gene could be detected in combination with other RALP genes and RALP-dependent adhesin genes in a strain-specific manner. The gene encoding the Nra regulator was predominantly found in opacity factor (OF)-negative serotypes. When analysing a rofA mutant in a serotype M2 strain, the strain specificity was also found in the positive and negative regulatory functions of RALP genes as well as in the type and number of virulence genes and functions controlled by the RALP genes. Of 17 virulence-associated genes tested, only one, the putative streptolysin S gene, was observed to be derepressed in RALP mutants of three different GAS serotype strains. This strain-specific variability of RALP regulon sizes is associated with different patterns of host cell attachment and internalization. In addition, RofA2 was shown to control expression of the ribosomal protein gene rpsL. As a consequence, it was demonstrated for the first time in streptococci that aminoglycoside resistance mediated by rpsL expression is apparently controlled by a virulence gene regulator.

Escherichia coli were recovered from the members of two two-person households and their associated septic tanks. The E. coli were isolated using selective and non-selective isolation techniques, characterized using the method of multi-locus enzyme electrophoresis and screened for the presence of virulence factors associated with extra-intestinal disease by using PCR. The growth rate–temperature relationships of strains from the two habitats were also determined. Temporal variation explained 25% of the observed electrophoretic type (ET) diversity in the humans. Among-host variation accounted for 29% of the observed allelic diversity. In one household, ET diversity of the E. coli population in the septic tank was significantly lower than ET diversity in the humans providing the inputs to the septic tank. Molecular analysis of variance revealed that, on average, strains recovered from the septic tank of this household were genetically distinct from strains recovered from the humans providing the faecal inputs to the septic tank. Further, the growth rate–temperature response of strains differed between strains isolated from the septic tank and strains isolated from the humans. Septic tank isolates grew better at low temperatures than strains isolated from humans, but more slowly at high temperatures compared to the human isolates. By contrast, no real differences in ET diversity, allelic diversity, or the growth charcteristics of strains could be detected between strains from the humans and strains from the septic tank of the other household. The results of this study suggest there are strains of E. coli that are better ‘adapted’ to conditions found in the external environment compared to strains isolated from the gastrointestinal habitat. Further, the finding that the numerically dominant clones and clonal diversity in secondary habitats can differ substantially from those found in the source populations will confound efforts to identify the sources of faecal pollution in the environment.

The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

It was found that infection of Escherichia coli by bacteriophage λ is inhibited in the presence of certain bile salts and carbohydrates when cells are in the ’OFF’ state for production of the phase-variable cell surface protein antigen 43 (Ag43). The inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. Expression of the gene encoding Ag43 (agn43) from a plasmid or inactivation of the oxyR gene (encoding an activator of genes important for defence against oxidative stress) suppressed this inhibition. A mutation, rpoA341, in the gene encoding the α subunit of RNA polymerase also facilitated phage adsorption in the presence of bile salts and carbohydrates. The rpoA341 mutation promoted efficient production of Ag43 in a genetic background that would otherwise be in the ’OFF’ phase for expression of the agn43 gene. Analysis of a reporter gene fusion demonstrated that the promoter for the agn43 gene was more active in the rpoA341 mutant than in the otherwise isogenic rpoA + strain. The combined inhibitory action of bile salts and carbohydrates on phage adsorption and the abolition of this inhibition by production of Ag43 was not restricted to λ, as a similar phenomenon was observed for the coliphages P1 and T4.

The effect of detrimental conditions on bacterial motility in Escherichia coli was investigated. Expression profiling of mutant E. coli strains by DNA arrays and analysis of phenotypic traits demonstrated that motility and low-pH resistance are coordinately regulated. Analysis of transcriptional fusions suggests that bacterial motility in response to an acidic environment is mediated via the control by H-NS of flhDC expression. Moreover, the results suggested that the presence of an extended mRNA 5′ end and DNA topology are required in this process. Finally, the presence of a similar regulatory region in several Gram-negative bacteria implies that this mechanism is largely conserved.

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIAGlc, the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.

Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K d) for binding to cAMP were determined. The K d values were 1·6 μM for Vfr and 0·4 μM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.

The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli–mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette–Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in M. tuberculosis during macrophage infection, by real-time RT-PCR. Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.

After growth of Rhodococcus rhodochrous in Sauton’s medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3–4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ‘non-culturable’ cells of the ‘Academia’ strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton’s medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4–5 months post-inoculation, of a homogeneous population of ostensibly ‘non-culturable’ cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 105 organisms ml−1, and this value was further increased by one log using supernatant from an actively growing culture. Populations of ‘non-culturable’ cells of Mycobacterium tuberculosis were also obtained by the filtration of ‘clumpy’ cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ‘non-culturable’ cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ‘non-culturable’ bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

When Bacillus subtilis is subjected to phosphate starvation, the Pho and σB-dependent general stress regulons are activated to elicit, respectively, specific and non-specific responses to this nutrient-limitation stress. A set of isogenic mutants, with a β-galactosidase reporter gene transcriptionally fused to the inactivated target gene, was used to identify genes of unknown function that are induced or repressed under phosphate limitation. Nine phosphate-starvation-induced (psi) genes were identified: yhaX, yhbH, ykoL and yttP were regulated by the PhoP–PhoR two-component system responsible for controlling the expression of genes in the Pho regulon, while ywmG (renamed csbD), yheK, ykzA, ysnF and yvgO were dependent on the alternative sigma factor σB, which controls the expression of the general stress genes. Genes yhaX and yhbH are unique members of the Pho regulon, since they are phosphate-starvation induced via PhoP–PhoR from a sporulation-specific σE promoter or a promoter that requires the product of a σE-dependent gene. Null mutations in key regulatory genes phoR and sigB showed that the Pho and σB-dependent general stress regulons of Bacillus subtilis interact to modulate the levels at which each are activated.