- Volume 148, Issue 8, 2002

Gluconacetobacter diazotrophicus is an endophytic diazotroph of sugarcane which exhibits nitrogenase activity when growing in colonies on solid media. Nitrogenase activity of G. diazotrophicus colonies can adapt to changes in atmospheric partial pressure of oxygen (pO2). This paper investigates whether colony structure and the position of G. diazotrophicus cells in the colonies are components of the bacterium’s ability to maintain nitrogenase activity at a variety of atmospheric pO2 values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO2 of 2 and 20 kPa. Imaging of live, intact colonies by confocal laser scanning microscopy and of fixed, sectioned colonies by light microscopy revealed that at 2 kPa O2 the uppermost bacteria in the colony were very near the upper surface of the colony, while the uppermost bacteria of colonies cultured at 20 kPa O2 were positioned deeper in the mucilaginous matrix of the colony. Disruption of colony structure by physical manipulation or due to ‘slumping’ associated with colony development resulted in significant declines in nitrogenase activity. These results support the hypothesis that G. diazotrophicus utilizes the path-length of colony mucilage between the atmosphere and the bacteria to achieve a flux of O2 that maintains aerobic respiration while not inhibiting nitrogenase activity.

Fluorescence in situ hybridization (FISH) was used to analyse the community composition of a sequencing batch reactor (SBR) operating with aerobic–anaerobic cycling and fed acetate as its sole carbon source. Phosphorus was removed from the SBR microbiologically. Marked shifts in the community structure occurred as the phosphorus/carbon (P/C) ratio in the feed was changed. When the P/C ratio was shifted from 1:10 to 1:50, FISH analysis showed that the percentage of β-Proteobacteria fell from ca 77% of the total bacteria to ca 38%. This decrease in the β-Proteobacteria coincided with a reduction in both the proportions of the β-proteobacterial Rhodocyclus-related phosphorus-accumulating bacteria and the biomass phosphorus content. FISH/microautoradiography and FISH/poly β-hydroxyalkanoate (PHA) staining showed that the Rhodocyclus-related bacteria assimilated acetate and synthesized PHAs anaerobically, and that they accumulated phosphorus aerobically. No Acinetobacter spp. could be detected in any of the communities, casting further doubt on their role in phosphorus-removing activated sludge systems. As the feed P/C ratio decreased there was a corresponding increase in the proportion of α-Proteobacteria and, to a smaller extent, in the proportion of γ-Proteobacteria; both the α- and γ-Proteobacteria consisted mostly of tetrad-forming cocci, fitting the description of the so-called ‘G-bacteria’ morphotype. The change in the proportions of Proteobacteria present paralleled increases in the biomass glycogen content. Both the α- and β-proteobacterial ‘G-bacterial’ populations assimilated acetate and synthesized PHA anaerobically. The α-Proteobacteria are considered responsible for glycogen production in these SBR systems.

Most filamentous bacteria in biological nutrient removal (BNR) processes have not been identified beyond their morphotype and simple staining reactions. Furthermore, the majority of sludge filaments observed under the microscope do not hybridize to commonly used phylogenetic probes for well characterized bacterial phyla such as the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Specific 16S rRNA-targeted oligonucleotide probes were designed for the phylum Chloroflexi (green non-sulfur bacteria) and optimized for use in fluorescence in situ hybridization. Chloroflexi have been implicated in BNR systems by phylogenetic identification of filamentous bacteria isolated by micromanipulation from sludge and culture-independent molecular phylogenetic surveys. The predominant morphotype responding to the probes was filamentous and these filaments were generally abundant in 10 Australian full-scale and two laboratory-scale BNR samples examined. Filamentous bacteria responding to a subdivision 1 Chloroflexi probe were rare in the samples, whereas subdivision 3 Chloroflexi filaments were very common in some sludges. This is in direct contrast to results obtained from molecular phylogenetic surveys of BNR systems where most sludge 16S rDNA clones belong to subdivision 1 and only a few to subdivision 3. It is suggested that filamentous bacteria belonging to the Chloroflexi phylum account for a large fraction of phylogenetically uncharacterized filaments in BNR systems and are likely to be abundant in such systems on a global scale.

In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989) R33 . J Bacteriol 171, 837–846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5′-ATTCACGCTN2TGAT-3′). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3·5) microcosms that were exposed to 13CH3OH or 13CH4. Distinct 13C-labelled DNA (13C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the 13C-DNA fractions, suggesting certain AOB assimilated a significant proportion of 13CO2, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the 13CH3OH and 13CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the α-amylase family and showed the greatest homology to the Bacillus megaterium group of α-amylases. The amyA gene was expressed in E. coli as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 °C in 5% (w/v) NaCl at pH 7·5, with significant activity retained in the presence of up to 25% (w/v) NaCl. It had a specific activity of 22·32 U mg−1 and required NaCl and CaCl2 for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H. orenii AmyA.

A gene encoding sorbitol-6-phosphate dehydrogenase (SorF) belonging to the sorbose operon (sorFABCDG) has been characterized in Lactobacillus casei. Inactivation of this gene revealed the presence of another sorbitol-6-phosphate dehydrogenase that was induced by D-sorbitol (D-glucitol). The gene encoding this activity (gutF) has also been isolated, sequenced and disrupted. The sorbitol-6-phosphate dehydrogenase genes (sorF, gutF) were required for growth on L-sorbose and D-sorbitol, respectively. Biochemical and transcriptional analyses of the wild-type and mutant strains demonstrated that L-sorbose and D-sorbitol induced sorF and the gene encoding the sorbose operon activator (sorR), while the expression of gutF was only activated by D-sorbitol. Furthermore, these studies indirectly suggested that a common metabolite of the L-sorbose and D-sorbitol metabolic pathways (probably D-sorbitol 6-phosphate) would act as the effector of SorR. The same effector would also be the inducer of gutF, although the two pathways seem to be subject to distinct regulatory mechanisms.

Class IIa bacteriocins may be used as natural food preservatives, yet resistance development in the target organisms is still poorly understood. In this study, the understanding of class IIa resistance development in Listeria monocytogenes is extended, linking the seemingly diverging results previously reported. Eight resistant mutants having a high resistance level (at least a 103-fold increase in MIC), originating from five wild-type listerial strains, were independently isolated following exposure to four different class IIa bacteriocin-producing lactic acid bacteria (including pediocin PA-1 and leucocin A producers). Two of the mutants were isolated from food model systems (a saveloy-type sausage at 10 °C, and salmon juice at 5 °C). Northern blot analysis showed that the eight mutants all had increased expression of EIIBgl and a phospho-β-glucosidase homologue, both originating from putative β-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). However, disruption of these genes in a resistant mutant did not confer pediocin sensitivity. Comparative two-dimensional gel analysis of proteins isolated from mutant and wild-type strains showed that one spot was consistently missing in the gels from mutant strains. This spot corresponded to the MptA subunit of the mannose-specific PTS, , found only in the gels of wild-type strains. The mptACD operon was recently shown to be regulated by the σ54 transcription factor in conjunction with the activator ManR. Class IIa bacteriocin-resistant mutants having defined mutations in mpt or manR also exhibited the two diverging PTS expression changes. It is suggested here that high-level class IIa resistance in L. monocytogenes and at least some other Gram-positive bacteria is developed by one prevalent mechanism, irrespective of wild-type strain, class IIa bacteriocin, or the tested environmental conditions. The changes in expression of the β-glucoside-specific and the mannose-specific PTS are both influenced by this mechanism. The current understanding of the actual cause of class IIa resistance is discussed.

Vanadium has an antibacterial activity against Pseudomonas aeruginosa, especially under conditions of iron limitation. Some degree of resistance to V is inducible by prior exposure to the metal. One mutant (VS1) with a higher sensitivity to V was obtained by transposon mutagenesis of P. aeruginosa PA 59.20, a clinical isolate. This mutant had an insertion in a non-coding region, upstream of a cluster of four genes. Three of them show similarities to genes corresponding to known P. aeruginosa antibiotic efflux systems, including an efflux protein, a membrane fusion protein and an outer-membrane porin. This cluster was named mexGHI-opmD. By allelic exchange, three mutants, ncr (for non-coding region), mexI and opmD were constructed in P. aeruginosa PAO1. Next to V sensitivity, the ncr, mexI and opmD mutants also showed reduced production of elastase, rhamnolipids, pyocyanine, pyoverdine and had reduced swarming motility, phenotypes that are known to be regulated by quorum sensing. All wild-type phenotypes, including growth in the presence of V, were restored by complementation with the complete cluster. The production of N-acyl-homoserine lactones (AHLs) was detected using the Chromobacter violaceum bioassay. Total extracts from the three mutants failed to induce the production of violacein by C. violaceum, although AHLs were detected by TLC and C. violaceum overlay. Violacein production was restored by complementation with mexGHI-opmD. The opmD mutant grew very slowly in LB or CAA medium, indicating that OpmD has an important physiological function for the cell. In conclusion, it is believed that the MexGHI-OpmD pump is probably involved in AHL homeostasis in P. aeruginosa.

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB – in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme’s hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD600 fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore’s ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination: the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

Knowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete. The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes. Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities. Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides. Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine. Both phases were poorly active in cytidine uptake. Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil. Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism. Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process. It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.

The amino acid sequence of the signal transducer PII (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria. PII of P. marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942. Moreover, P. marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite. This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (Ci; K m,app 240 and 4 μM, respectively), a result consistent with the unphosphorylated form of PII acting as a sensor for the control of Ci acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803. The present data are discussed in relation to the genetic information provided by the P. marinus MED4 genome sequence.

Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the −10 region and a region immediately upstream of the −35 region of the σ70 promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the σ54-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaRΩKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed. In addition to this, phaR expression is subject to autoregulation. Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.

The entomopathogen Photorhabdus luminescens secretes many proteins during the late stages of insect larvae infection and during in vitro laboratory culture. The authors have previously characterized and purified a 55 kDa zinc metalloprotease, PrtA, from culture supernatants of P. luminescens. PrtA is secreted via a classical type I secretory pathway and is encoded within the operon prtA–inh–prtBCD. The 405 bp inh gene encodes a 14·8 kDa pre-protein that is translocated to the periplasm by the classical signal-peptide-dependent sec pathway, yielding the mature 11·9 kDa inhibitor Inh. Inh is a specific inhibitor of the protease PrtA. This study describes the purification of Inh and the initial characterization of its in vitro protease inhibition properties.